(PDF) [Advances in Protein Chemistry] Advances in Protein Chemistry Volume 10 Volume 10 || Metabolism of the Aromatic Amino Acids

Metabolism of the Aromatic Amino Acids

BY C. E. DALGLIESH Postgraduate Medical School, Ducane Road, London, England

CONTENTS Page

I. Introduction . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . , . . . . . . . . . , . . , 33 1. Historical . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . , . , 34 2. General . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . , . . , . . , , . . . . . 35 3. “Essentittl” N:tture of Aromatic Amino Acids for AIammaIs. . , , , , . . 35

11. Biosynthesis of the Aromatic Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . 36 1. Experiments Using Mutants of Rlicroorgnnisms. . . . . . . . . . . . . . . . . . . 36 2. The “Common-Precursor” Pathway of Aromatic Iliosynthesis.. . . . . . 36 3 . Final Stages in Phenylalanine Biosynthesis . . . . . . . . . . . . . . . . , . . , 39

5. Final Stages in Tryptophan Biosynthesis . . , . . . . . . . . . . . . . . . . 40 6 . The “Straight-Chain” Pathway of Aromatic Biosynthesis.. . . . . . . . . . . 42 7 . Isotopic Evidence on the Pathwtys of Sromatic Amino Acid I3iosy1i-

thesis.. . . . . . , . . . . . . . . . . . . . . . . . . . , . . , . 43 111. Degradation of Phenylalanine and Tyrosine to Acetoacet,nte; the Principal

Route Used by Mammals. . , . . . . . . . . , . . . . . . . . . . 40

4. Final Stages in Tyrosine Biosynthesis . . . . . . . . . . . . . . . 40

. . . . . . . . . . . . . . . . . , .

111.4. Evidence Derived from Inborn Errors of Metabolism. . . . . . . . 46 . . . . . 47

tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 52 55

1. Allcaptonuria, and Related Work on Man and 1nt:iot Anirnals 2 . Tyrosinosis and Other Cases of p-Hydrosyphenylpyruvic Acid 1Sxcre-

3. Phenylketonuria (Oligophrenia I’henylpyruvica; Falling’s Syndrome). , 11113. Enzymic Experiments on the Normal Pathway in Mammals.. . . , . , . . , . .

1. General Outline of the l’at,hway.. . . . . . . . . . . . . . . . . . 55 2. Conversion of Phcny1:ilanine to Tyrosine. . . . . . . . . . . . . 5s 3 . Conversion of Tyrosine t.o p - I I ~ t l r o s . p h e n ~ l p y r ~ ~ v i c Acid . . . . . . . . . . . 4 . Conversion of p-Hydrox!.phcnylpa,ruvic Acid to 2,5-l)ihytlros~;phcnyl-

pyruvic arid hom*ogentisic acids. E’unction of Ascorllic Acid and of Hematopoietic Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

5 . Conversion of hom*ogeritisic Acid t o YIaleylacetoacet,ic, Fumarylaceto- acetic, Fumaric, and Acetoncet,ic Acids.. . . . . . . . . . . . . . . . . . . . . . 64

IV. Tyrosine Degradation by t,he Catechol P a t h w i y . . . . , . . . . . 65 1. Adrenaline, Noriidrenaline, and Thcir Biogenesis . . . . . . , . 66 2 . Metabolic Degradation of Noradrenaline and Adrenaline. Adreno-

chrome. . . . . . 68 3 . Alelanogenesis and Alhiriism. . . . . . . . , . , , . . . . . . . , 0n -1. T l i ~ Ciiiecliol h t h w n y in thcb I n n c ~ ~ t . . . . . . . . . . . . . . . . 71

3!)

\‘. Tyrosiire ;\Iet:il~olism via Thyroid Hormones :ind Ot,lic:r Halogeniited De- , . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . .

rivatives, , . , . , . , . . . . . . . . . . . . . 71

32 C . E . DALGLIESH

1’:tge 71 74 75 75

by Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 1 . Products Based on Decarboxylation and Amine Oxidation 2 . Products Based on Oxidative Deamination or Transamination. . . . . . . 77 3 . Phenol Formation from Tyrosine . p-Tyrosinase . . . . . . . . . . . . . . . . . . 78 4 . Degradations Involving Opening of the Aromatic Ring . . . . . . . . . . . . . . 78

VII . Tryptophan Degradation by the Kynurenine-Nicotinic Acid 1’at.hway . . . 70 1 . Establishment. of the Relation between Tryptophan and Nicotinic

Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 2 . The General Outline of the Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 3 . The Conversion of Tryptophan to Formylkynurenine and Kynurenine . 83 4 . Tryptophan Pcroxidase-Oxidase Adaptation . . . . . . . . . . . . . . . . . . . . . . . 85 5 . Conversion of Kynurenine to Hydroxykynurenine . Role of Ribo-

flavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 0 . Iiydroxykynureninc and Insect Eye Pigments . . . . . . . . . . . . . . . . . . . . . 87 7 . Kynureninase, Kynurenine Transaminase, and the Formation of

Anthranilic, Kynurenic, Hydroxyanthranilic., and Xanthurenic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

8 . Mechanism of Action of Pyridoxal Phosphate in Reactions Involving Aromatic Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !)1

0 . Excretion of Hydroxykynurenine and Xanthurenic Acid by Man . . . . 04 10 . Side Reactions of Kynurenine, Hydroxykynurenine, Anthrariilic Acid,

and Hydrosyanthranilic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !)5 11 . Conversion of Hydroxyanthranilic Acid to Sicotiriic Acid . . . . . . . . 97 12 . Tryptophan, Nicotinic Acid, and the Pyridine Sucleotides . . . . . . . . . . 100 13 . Further Metabolism of Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . 101

VIII . Tryptophan Degrttdrttion by the Enl.eramin e-Serotonin Pathway . . . . . . . . 103 1 . Biosynthesis of 5-IIydroxytryptamine . . . . . . . . . . . . . . . . . . . . . 104 2 . Degradation of 5-Hydroxytryptamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3 . N-Methylated Derivatives of 5-Hydroxytryptamine . . . . . . . . . . . . . . . . . . 107

I X . Routes for Tryptophan Degradation Used Principally by Microorganisms . 108 1 . Urinary Indolcscetic Acid and Indoleuceturic Acid . Urorosein . . . . . 700 2 . Bacterial Degrttdat. ion via Indole . The Tryptophnnrtsc Reaction . . . 110 3 . Fiirthcr Degradation of Indole by Hacteria . . . . . . . . . . . . . . . . . . . 111 4 . Origin of IJrinitry Indican, Iudigo, Indirubin, Bkat. oxyl :tnd Skatole Red 111

X . Tryptoph:in Metabolism in Plants . Heteroauxin . . . . . . . . . . . . . . . . . . 113 1 . Riogenesis a r i d Ilegradntion of Indoleacetic -4rid i n Plant8 . . . . . . . . . . . 114 2 . Other Jnclolic €’l:int Growt Ii 1Iormont.s . . . . . . . . . . . 1 I4

115 1 . Probably 1lel:itcd hIet:tt)olic Products in Microorganisms . . 115 2 . Probably Relat.et1 Metabolic Products in Plants and Fungi . . . l l f i 3 . Flnvorioids and I, ignin . . . . . . . . . . . . . I l(j 4 Alkaloids 117

X I 1 . Future Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 XI11 . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

1 . Thyroxine and It. s Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Triiodothyroninc and Its Biogenesis . . . . . . . . . . . . . . . . . . . . . . . 3 . Metabolic Fate of the Thyroid Hormones . . . . . . . . . . . . . . . . . . . . . . 4 . Other Naturally Occurring Halogenated Tyrosines . . . . . . . . . . . . . . . . . . .

V I . Pathways of Phenylalanine and Tyrosine Metabolism Utilized Principally

XI . N:iiiiral Products I’rolxthly Related to the Aromatic Amino k i d s . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

METABOLISM O F THE AROMATIC AMINO ACIDS 33

I. INTRODUCTION I’henylalanine,1 tyrosine, and tryptophaii are unique among the amino

acids for the wide variety of reactions which they undergo.

Phenglalanine Tyrosine o7 CH,.YH*COIH

Try p top hail

SHZ H

Besides being fundamental constituents of proteins they are the parent substances from which powerful hormones are derived, for example, adrenaline (epinephrine), noradrenaline (norepinephrine), thyroxine and related substances, 5-hydroxytryptamine (enteramine, serotonin), and the plant hormone indoleacetic acid. Tryptophan is also the precursor of the B vitamin nicotinic acid and hence of part of the important pyridine nucleotides. All three aromatic amino acids are potential precursors of other substances having powerful physiological activity, for example, many of the alkaloids. Errors in the metabolism of the aromatic amino acids in man can give rise to sometimes serious, but fortunately comparatively rare, disorders such as alkaptonuria and phenylketonuria. The numerous metabolic pathways involved in aromatic amino acid metabolism therefore make an important as well as an interesting study.

Protein ingested by a living organism in general undergoes proteolysis, the liberated amino acids joining the respective amino acid “pools” of the organism. Simultaneously the organism’s own protein is being broken down and the resultant amino acids also join the amino acid “pools” together with newly formed amino acid molecules in those organisms in which biosynthesis occurs. Once amino acid molecules from these various sources have entered the “pools” they are indistinguishable (e.g., 614). From these pools material is withdrawn for ( I ) synthesis of new protein and (2) degradation or other metabolic transformations. I n the fully grown organism in the steady state as much amino acid must be metabolized as is ingested and/or biosynthesized (though not, of course, necessarily the identical molecules). This review covers these metabolic degradations and transformations and also biosynthesis. The incorporation of the aromatic amino acids into proteins will not be considered.

Throughout this review i t is to be assumed unless otherwise stated that amino acids are of the L-configuration.

34 C. E. DALGLIESH

Many thousaiids of papers have hcen written on the subject of this rc- \.iew, of which only a sinall fraction tan Ite cited here. The aim has been to give some idea of the historical development of our knowledge, followed by a detailed statenient of the present state of that knowledge. The main em- phasis has been laid on those pathways which appear to account for the greater part of normal aromatic amino acid metabolism in man; subsidiary pathways are dealt with more briefly. An attempt has been made to cover relevant papers received in England up to the end of 1954.

1. Historical

Tyrosine was the first of the aromatic amino acids to be discovered, iii 1846, by Liebig (558), who obtained it on acidifying and cooling a solution of casein which had been fused with potash. Better methods of hydrolysis than alkali fusion were soon discovered, and tyrosine was isolated from numerous other sources. The relative insolubility of tyrosine (0.048 g./100 ml. water a t 25°C. as compared with corresponding values of about 3 g./100 ml. for phenylalanine, 1.14 g./100 ml. for tryptophan, and 25.3 g./100 ml. for glycine) causes tyrosine, often mixed with cystine (solubility 0.01 1 g./ 100 ml. a t 25”C.), to separate particularly readily from protein hydrolyzates and also, in certain pathological states, from body fluids. The correct structure of tyrosine was first suggested by Barth (32), was strongly supported by Baumann (36), and was finally proved by synthesis by Erlen- meyer and co-workers (227, 229). Emil Fischer was the first to resolve synthetic m-tyrosine into its optical isomers (266).

Phenylalanine was first obtained by Schultze and Barbieri in 1879 (779, 780) from etiolated lupine sprouts. Its structure was shown (781) by comparison with synthetic material prepared independently by Erlenmeyer and co-workers in 1882 (228). Synthetic DL-phenylalanine was first optically resolved in 1900 (267). The difficulty of isolating phenylalanine from protein hydrolyzates prevented recognition of its widespread occurrence in proteins until after Emil Fischer had developed his method for separation of the amino acids by fractional distillation of their esters.

Tryptophan was first isolated only a t the beginning of this century (411). A number of color reactions of proteins were extensively studied in the latter half of last century and numerous attempts were made to isolate the chromogen responsible. The name “tryptophan” was given to this chromogen in 1890 by Neumeister (645). The chromogen was soon associated with the substance giving rise to indole on bacterial putrefaction of proteins. The failure of many early attempts to isolate tryptophan was probably due to the fact that it is destroyed on acid hydrolysis. The successful isolation by Hopkins and Cole (411) used enzymic hydrolysis of casein, but the chief reasons for their success were their discovery of mercury salts as

METABOLISM OF THE AROMATIC AMINO ACIDS 35

precipitating agents for tryptophan and their prior development (410) of a more sensitive version of the original Adamkiewicz reaction (2, 3) with which they could follow their isolation procedure. The structure of tryptophan was the subject of much discussion but mas finally settled in 1907 by the synthesis of Ellinger and Flainand (221).

This discussion has surnniniized only the priticipal laiidinarks in the early history of the aromatic arniiio acids. For a complete account the excellent review by Trickery and Schmidt (894) should be consulted.

2. General

The geiierd chemical and physicochemical properties of the aromatic amino acids are outside the scope of this review, but attention is called to the following prime sources of information: synthesis and criteria of purity (207) ; stereochemistry and optical properties (640) ; physicochemical behavior (152).

3. “Essential” Nature of Aromatic Amino Acids for Mammals

For higher organisms, e.g., maninials, phenylalanine and tryptophan are so-called “essential” amino acids, i.e., they cannot be synthesized by the animal and must be supplied in the diet (for man, see 728, 729). Tyrosine is derived, as we shall see later, from phenylalanine, and is not therefore itself an essential amino acid as long as the phenylalanine intake is adequate (726, 950) ; if the conversion of pheiiylalanine to tyrosine is inhibited, as in phenylketonuria, tyrosine can become essential (65). The “essential” nature of the aromatic amino acids is a reflection of the general inability of higher organisms to synthesize the benzene ring.

For the requirements of phenylalanine and tryptophan by man and the rat, see 14, 15a, 153,630, 631, 653,728,729,732a1 732b, 750,757a1 and the review by Albanese (6).

For example D- phenylalanine is used by rat, mouse, and man (15, 35, 727, 730, 962), whereas D-tryptophan is used by the rat (53, 54, 759, 895), is partially used by the mouse and pig (139,867), and is not used by man (7,29). The uti- lization of the D-amino acids is probably determined by the relative rates of absorption of the D-amino acid from the intestine, and of conversion of D- to L-amino acid in the liver (288). The conversion of D- to L-phenylalanine is reduced in vitamin-B6 deficiency (52) , as is to be expected for a transformation involving transamination to phenylpyruvic acid. Phenylpyruvic and indolepyruvic acids, the a-keto acids corresponding to phenylalanine and tryptophan, may also, to an extent varying with the species, satisfy growth requirements (e.g., 55, 109,436, 725, 911).

&Amino acids vary in availability with the species.

36 C. E. DALGLIESH

11. BIOSYNTHESIS OF THE AROMATIC AMINO ACIDS

Biosynthesis of the carbon skeleton of the aromatic amino acids is coii- fined to the lower organisms. Plant material is the main ultimate source of the aromatic amino acids for animals and man, though some may also derive from bacteria, cr.g., in the gut. Sumerous theories have been advanced in the past as to the origin of aromatic rings (e.g., 707), but oiily rcceritly has definite cviderirc beeii forthcoming, derived from work with micro- organisma. There o nay well be a similar biosynthetic pathway iu plants, but there is its yet little direct evidence oil this point. Two main techniques have been uscd. Davis and his school have studied the requirements and excretion products of mutants of microorganisms, aiid several workers have studied biosynthesis from labeled precursors using isotope techniques.

1 . Experiments Using Mictants of JIIicrooryaniswu

Mutants of microorganisms can iiow be conveniently produced arid isolated from parent wild-type strains (for review, see 186). Such mutants are invaluable for the determination of metabolic sequences. If, for example, there occurs a sequence

A - + B + C - + D . . . + X in which X is necessary for growth of the organism, then a mutant blocked in the coiiversiori of I3 to C will not in general grow unless C, or some suh- stance subsequent to it iu the metabolic chain, is supplied to the organisin. Similarly 13, or a derivative, may accumulate in the culture medium, especially if A is supplied. Many complications occur owing to alteriiative pathways, ((leakage” due to incomplete blocks, and competitive inhibition of one metabolite by another close to it in the chain (e.g., 187-189). Sever- theless simple relationships exist in a sufficient number of cases to make the method a most fruitful one.

2. The “Common-Precursor” Pathway of Aromatic Biosynthesis

Davis (184) selected a large iiuinbcr of mutants of Escherichia coli requiring two or more aromatic amino acids for growth, and then tested a large number of substances to see if any could relieve the growth inhibition. Success was attained with shikimic acid (215,268), at that time a relatively obscure natural product, This indicated either that shikimic acid (structure diagram 1) was a true aromatic precursor or that it could readily be transformed into a true precursor. The likelihood that shikimic acid was a true precursor was increased when other mutants were found to accumulate shikimic acid in the medium, from which it could be isolated (184, 185).

Not all the mutants responding to shikimic acid responded to a mixture of

METABOLISM OF THE AROMATIC AMINO ACIDS 37

the aromatic amino acids. Some of these, however, responded when p- aminokmzoic acid was also supplied. The biosynthesis of p-aminohenzoic acid is therefore obviously related, a t least in E. coli, with biosynthesis of the aromatic amino acids. In further mutants a mixture of the aromatic amino acids and p-aminobenzoic acid still did not produce growth, unless supplemented with a trace of shikimic acid or with a culture filtrate of wild- type organisms. These mutants therefore required still another substance related to aromatic biosynthesis. Extracts of wild-type organisms were examined by paper chromatography, and it soon became clear that paper itself contained the unknown factor. A study of likely trace contaniinants of paper finally led to the identification of this bacterial growth factor as p-hydroxybenzoic acid (183). Evidence was also obtained that under certain conditions of pH, still another growth factor, called the sixth factor, could become necessary (e.g., 185, 188). This has not yet been identified.

Davis concluded that shikimic acid was a common precursor of phenylalanine, tyrosine, tryptophan, p-aminobenzoic acid, p-hydroxybenzoic acid, and an unknown sixth factor, and he next set out to determine other substances lying on the biosynthetic pathway. The various mutants were therefore tested for syntrophism, i.e., for the ability of one mutant to produce a substance necessary for the growth of another mutant. There was thus found a thermolabile substance, X, which was a true precursor of shikimic acid (184). X was isolated from culture filtrates and identified as 5-dehydroshikimic acid (744). Similar experiments revealed a substance, W, which was a true precursor of substance X (187, 193). This also was isolated and shown to be 5-dehydroquinic acid (906). The enzyme, named 5-dehydroquinase1 converting dehydroquinic acid to dehydroshikimic acid has been partially purified (606). It is fairly stable, has a high specificity, appears to have no cofactors, and is of wide occurrence in bacteria, algae, yeasts, and plants but, as expected, could not be found in mammalian liver.

The enzyme, 5-dehydroshikimic reductase, converting dehydroshikimic to shikimic acid, has also been studied (964a). It requires triphosphopyri- dinenucleotide as cofaetor .

These results are summarized in diagram 1. Substance CP represents a common precursor which has not yet been identified but the existence of which is shown by the existence of mutants which require all six aromatic factors but excrete shikimic acid into the medium.

Study of mutants with double, up to quintuple, requirements has shown (187) that the growth requirements for the various factors are always satisfied in the following order; p-hydroxybenzoic acid, p-aminobenzoic acid, tryptophan, and finally phenylalanine and tyrosine. This order parallels the quantita-

The six aromatic factors are not all synthesized equally readily.

38 C. E. DALGLIESII

I I *

Phenylalanine Tryptophan

Glucose

1 p-Hydroxybenzoic acid

110% COzH I10 COZH o..L,Il 0 -Qo,, 0 IIO..’

J$ 0 JOH x z x z 7 stnge blucked €10

Quinic dehydrogena3e

O H OH 5-Dehydroquinic acid Quinic acid

5-Dehydroquinnse

c ( ):I 1 Side rcactiun

nhich mag

OH OH 5-Dehydroshikimic acid Protocatcchuic acid

Dehydroaliikimic reductnse I

YOzH

Compound 21 I I I I I I I

I ? I

HO-’ OH OH \

Shikimic acid Phosphoshikimic acid

CP (common precursor)

tive growth requirements, the amounts, on a molar basis, of p-hydroxybenzoic and p-aminobenzoic acids required for growth being about one- thousandth of the requirement for tryptophan, which in turn is about one-quarter the requirement for pheriylalanine or tyrosine.

The further metabolic changes undergone by shikimic acid are not yet clear. Mutants have been found that excrete two derivatives of shikimic acid (192). One of these, Z1, tentatively identified as a cyclic acetal of shikimic acid with pyruvic acid (192), may be a precursor of prephenic acid (191a). The other, 22, is a phosphoshikimic acid, and possibly, but not yet certainly, is a normal metabolite succeeding shikimic acid in the chain.

METABOLISM OF THE AROMATIC AMINO ACIDS 39

Similarly the immediate precursors of dehydroquinic acid, which must include open-chain compounds, are not yet known. Earlier work (301) with Neurospora suggested that quinic acid might be a precursor. How- ever, quinic acid did not support growth of any of the E. coli mutants, though it did support growth of some Aerobacter mutants. It was established (193, 607) that quinic acid is not a true aromatic precursor, but that some organisms have the ability to convert it, as shown in the diagram, into dehydroquinic acid, which is a true precursor. The biosynthetic pathway in Neurospora appears to be the same as in E. coli (856). A Neurospora mutant blocked in the conversion of dehydroshikimic acid to shikimic acid converted a large part of the dehydroshikimic acid to protocatechuic (3,4-dihydroxybenzoic) acid (856; cf. diagram 1).

The ultimate derivation from glucose, shown in diagram 1, is considered in the section on isotopic evidence. A combination of mutant, enzymic, and isotopic techniques suggests that sedoheptulose-1 ,7-diphosphate is an intermediate in the conversion (456a,b, 823a).

3. Final Stages in Phenylalanine Biosynthesis

Experiments with mutants have also revealed the final stages of phenylalanine biosynthesis. Both Davis (190) and Japanese workers (459) obtained mutants excreting a labile substance, subsequently called prephenic acid, which was very readily converted into a second substance, Y, which was in turn converted into phenylalanine. Y was identified as phenylpyruvic acid (190). Prephenic acid was isolated (907) and its structure (see diagram 2) demonstrated (cf. 288a).

H02C CHz*CO*CO,H CHz CO * COJI CP Common Ciihiioao precursor stages

I OH

Prepheiiic acid l ’ l~enyl~~yruvic acid

Phenylalanine Diagram 2. Final stages of phenylalanine biosynthesis in E. coli.

Hnddox (333), working with Neurosp*rn mutants, llns suggested that in this organism a-phciiylglycine is closely coiinected with phenylalanine biosynthesis. It was not possible to establish whether it mas a true precursor

40 C. E. DALGLIESH

or only a product of “shunt” metabolism formed from a true precursor, and more complete identification is in any case desirable.

4. Final Stages in Tyrosine Biosynthesis

Some microorganisms may resemble the higher organisms in being able to convert phenylalanine directly to tyrosine; thus it can occur in Vibrio (167) and Pseudomonas (605) and has been claimed for E. coli (48; but cf. 807). However in Lacfobacillus arabinosus tyrosine is formed by a route not involving phenylalanine (20), as is apparently also the case in Aero- bacter aerogenes (605). The direct conversion of phenylalanine to tyrosine is claimed by advocates of the “straight-chain” pathway of aromatic biosynthesis described later.

Otherwise no immediate precursors of tyrosine appear t o have been reported. Transamination of p-hydroxyphenylpyruvic acid has been suggested to be the final stage in yeasts (474), and may occur in E. coli (809), and isotopic evidence, discussed later, suggests that even if tyrosine is not formed from phenylalanine, the method of introduction of the tyrosine side chain is very similar t,o that postulated above for phenylalanine. Forma- tion of p-hydroxyphenylpyruvic acid from prephenic acid can be readily visualized.

6. Final Stages i n Tryptophan Biosynthesis

Anthranilic acid and indole are precursors of tryptophan in numerous microorganisms and fungi (e.g., 5, 263, 264, 602, 741, 783, 785, 816, 854, 855, 876), and i t is probable that anthranilic acid is derived, with intermediate steps, from the common precursor, CP of diagram 1. The conversion of anthranilic acid to indole and tryptophan has been shown unambiguously in Neurospora with the use of isotopic techniques (93, 663). There may, however, be other pathways for tryptophan biosynthesis (45, 702). Tryptophan can, for example, be formed by transamination of indolepyruvic acid (e.g., 470, 912), which might be formed other than via aiithranilic acid. Thus aromatic-requiring mutants have been found which accumulate unidentified indole compounds (307).

A i i t h ~ ~ i i l i ~ . acid Ill~lolt-

Diagram 3. Final stages in tryptophan biosynthesis.

llaskins and Mitchell (365) foulid that a it’eiirosporu iiiutaiit blocked IJO- tween anthranilic acid and tryptophan accumulated aiithranilic acid when

METABOLISM OF THE AROMATIC AMINO ACIDS 41

grown on a high-tryptophan medium. They therefore postulated a “tryptophan cycle”:

Tryptophan + kynurenine + hydroxykynurenine, etc.

T I indole + anthranilic acid

Partridge and co-workers (663) showed that such a cycle plays no part under conditions of normal tryptophan synthesis, and Adelberg (4) has plausibly ascribed the original results to adaptive formation of the enzyme kynureninase (discussed in detail later) induced by the high tryptophan content of the medium.

In the conversion of anthranilic acid to indole it has been found that in Neurospora the carhoxyl group is lost (652), whereas in R. coli it has been claimed that the carboxyl group is retained and the additional carbon atom required is supplied by methionine (56). However, Yanofsky has prepared from E. coli a cell-free system carrying out almost quantitatively the conversion of anthranilic acid to indole. This system requires adenosine triphosphate (ATP) and ribose-5-phosphate or ribose or, in certain circum- stances, hexose diphosphate or hydroxypyruvic acid in place of the ribose (967). Tracer experiments showed that the carboxyl group was lost in the coilversion (967) so that a two-carbon (or higher) unit must be added, and it has been shown that this unit becomes attached to the benzene ring a t the same position as the displaced carboxyl group, aniline not being an intermediate (969). Acetate may be the unit involved, becoming attached to the benzene ring through the methyl group (686a,b). Harley-Mason (361a) has realized a similar conversion chemically.

The conversion of indole to tryptophan has been much more extensively studied. This is brought about by direct reaction of indole and serine under the influence of the enzyme tryptophan desmolase (better named tryptophan synthetase) (302, 853,854) which requires pyridoxal phosphate as a coenzyme (890). The enzyme has been obtained in the cell-free state (890) and partially purified (965) and its genetics in Neurospora studied in detail (966). Zinc appears to play some part in tryptophan desmolase formation or function (628).

The mechanism of function of pyridosal phosphate in tryptophan biosynthesis is considered in more detail later in discussing other pyridoxine- dcpeudeiit cnzyrnes. . t i i activated fornl of serinc is formed which reacts with indole. I>ehyclration call take place in two ways: intermolecularly, involving loss of water between the p-hydroxyl of activated serine and the 0-hydrogen of indole, or intramolecularly, involving loss of water from ac- t ivated seriiie to give activated aminoacrylic acid, which theri adds on to the P-position of indole. Tatum and Shemin (858) in ingenious experiments

42 C. E. DALGLIESH

involving triply labeled (D, N”, p-C”) serine, concluded that the latter mechanism is more probable, but did not distinguish between the possible tautomeric intermediates.

6. The “Straight-Chain” Pathway of Aromatic Biosplhesis

Though the evidence for the “common-precursor” pathway is weighty, this pathway has not been universally accepted as the sole route for aromatic biosynthesis. An alternative “straight-chain” pathway has been proposed, largely based on evideiice obtained with competitive inhibitors of the aromatic amino acids such as phenylserine and fluorophenylalanine. Beerstecher and Shive (47) originally suggested that in 23. coli tryptophan could be directly converted to phenylalanine. Bergmann and co-workers (57, 58) studied the question in greater detail both by use of analogue inhibitors and by studying the “sparing effect” of one aromatic amino acid on the requirement for the others. They deduced the metabolic scheme shown in diagram 4, the dotted lines representing alternative pathways :

precursor

tryptophan -+ phenylalanine - tyrosine + metabolic product of tyrosine -3

~ - - - - - - - - - - - - - - 1 t - - - - - - - - - - - 3

Diagram 4. “Straight-chain” pathway proposed for aromatic amino acid biosynthesis.

The possibility does not appear to have been sufficiently coilsidered that apparent conversion might be due to reversible transformations to a common precursor (cf. 187). Evidence obtained from inhibition studies is suggestive rather than conclusive, and other interpretations are possible; for example, Pimmonds ct al. (808), studying phenylserine inhibition in B. coli mutants, concluded that the inhibition produced by pheriylserine mas iii- hibition of use of phenylalanine for growth, and not of phciiylalanint. tksyiithesis, aiid they wnsidered that their results supported tlic (‘coin- nion-precursor” rather than the “straight-chain” pathway. S y c ri al. have suggested (650) that in Neurospora phenylalaniiie and tyrosine arc precursors of tryptophan, and 13eerstecher has suggested (45) that) in lactic acid bacteria phenylalanine can be converted to tryptophan.

Until more evidence is forthcoming the direct transformation of t ryptophan to phenylalanine implied in the “straight-chain” pathway should not be considered as finally established. The observation (458) that the inhibition of l d . coli by azaserinr can be reversed by tryptophan, I,hciiylnlaiiiii~., or tyrosiiicl miy Ic:~d to future useful rcsults.

METABOLISM OF THE AROMATIC AMINO ACIDS 43

7. Isotopic Bvidencc on thc Pathways of Aromatic A m i n o Acid Biosynthesis

Ehrensviird and co-xvorkers (25) grew the yeast Torutopsis utilis on, as sole carbon source, acetate labeled with C14 in the carboxyl carbon and C13 in the methyl carbon. They then isolated and degraded the tyrosine and studied the distribution of activity in various positions. Acetate carboxyl contributed to the tyrosine carboxyl and to C-4 of the aromatic ring, whereas the a- and &carbon atoms of the side chain and carbons 1, 2, and C, of the ring came predominantly from the acetate methyl carbon. This result immediately excluded cyclization of a straight chain of fatty acid type in which alternate carbon atoms should derive from the acetate methyl and carboxyl. It was suggested that the most plausible explanation of (cf. also 804) was that the aromatic ring was in some way connected with triose- hexose systems. Gilvarg and Bloch (289, 290) grew the yeast Saccharo- myces cerevesiae on a mixture of either unlabeled glucose and labeled ( l-Ci3, 2 G 4 ) acetate, or on labeled (l-Ci4) glucose and unlabeled acetate. In the presence of glucose no incorporatlon of labeled acetate occurred into phenylalanine or tyrosine, though there was extensive labeling of glutamic acid, nspartic acid, leucine, and lysine. Glucose was therefore the aromatic precursor, and not acetate or metabolically related substances such as ketoglutarate or oxalacetate. When (l-C14) glucose was substrate the label appeared in both ring and side chain of phenylalanine and tyrosine. On degradation none of the label appeared in carbons 1,3, or 5 of the ring, whereas the greater part occurred in carbons 2 and/or G (carbons 2 and 6 were not distinguishable by the degradative procedure). These results made it unlikely that direct cyclization of glucose had occurred, and ruled out condensation of two isotopically equilibrated three-carbon units which should give labeling in ortho or para positions thus:

I I c c c’ c*

C’ Head-to-tail union of

&carbon unlts resulting in p a w labeling

Tail-to-tail union of 3-carbon units resulting

In orlho labeling

More extensive studies (291) on biosynthesis from ( l-C14) glucose, using improved procedures for degradation and location of the activity, revealed high activity only in the /3-carbon of the side chain and in carbons 2 and/or G of the ring, confirming the previous results. The direct cyclization of glucose still remained a possibility; this should give rise to label in one position only, but it could not be determined whether activity appeared in both

44 C. E. DALGLIESH

carbons 2 and 6 or in only one of these. Direct cyclization was, howcvoi~, considered unlikely, and i t was suggested (cf. also 211) that there might, o(*- cur a condensation of a triose with a tetrose to give sedoheptulose or sorw other seven-carbon sugar.

Ehrcnsvard and lteio (213) grew both h'. coli and Neurospora crassa O I I

(l-CY4, acetate. Both these organisms used the same mechanism for aromatic biosynthesis, which was similar to that in yeasts. Degradation of tyrosine showed that the ring could not have been formed by direct cyclization of glucose, and these authors favored a scheme involving co11- densation of erythrose and triose in presence of aldolase (cj'., e.g., 41 5, 861 ), or less probably, a C6-G union. This scheme can be represented as follow, M representing a carbon derived from acetate methyl and C, from awtate carboxyl :

C /

FC &I-c -If '0 - \ / C-C

M + c-c \ "": c--c 1

or I - I

Acetate Glucose C, and C3 fragments lieptuloses etc.

Two possibilities were considered for the origin of the side chain, ( a ) direct addition of a two-carbon unit to a cyclized seven-carbon unit, or ( b ) addition of a three-carbon unit to the seven-carbon unit with expulsion of the one-carbon side chain:

\ + c-c-c pp--c Ehrensvard and Reio favored alternative (a ) . Alternative ( b ) strik-

ingly resembles the mechanism of phenylalanine biosynthesis revealed hy mutant techniques (see p. 39) ; that this mechanism can apply to tyrosine was rendered possible by work of Thomas and co-workers and was rendered extremely probable by work of Sprinson and co-workers.

Thomas P t al. (866) degraded yeast grown either aerobically or anaero- bically on carboxyl-labeled acetate or carbonyl-labeled pyruvate. In aerobic pyruvate cultures the side chain of tyrosine was in all probability de-

METABOLISM OF THE AROMATIC AMINO ACIDS 45

rived from an intact pyruvate molecule (cf. also 166). The distribution patterns of activity supported conclusions of earlier workers (1) that direct cyclization of glucose formed from pyruvate did not occur, and (2) that none of the intermediates involved could have been in equilibrium with symmetrical products of glycolysis or the Krebs cycle. (Krebs cycle intermediates can, however, cause an increase in tyrosine biosynthesis; 473, 475.) Spriiisori i t al. (824) have settled thc origin of the side chain in. E. coli by growing the organism on a mixture of unlabeled glucose and labeled shikimate, and subsequently isolating and degrading the tyrosine. -4s required by alternative (b) above, the carhosyl of shikimate had been eliminated and, as carbons 2 and 6 of shikimate corresponded to carbons 2 and 6 of the ring of tyrosine, the side chain of tyrosine must have entered the same position in the ring that was occupied by the shikimate carboxyl. Further- more it was shown that the side chain, in contrast to the ring, could have been in equilibrium with three-carbon glycolytic intermediates.

The above results make i t clear that glucose should be regarded as the precursor of the aromatic amino acids. In an attempt to elucidate the steps in this conversion Sprinson and co-workers have therefore combined isotope and mutant techniques. Using a mutant of E. coli accumulating shikimic acid in the medium, labeled bicarbonate, formate, and acetate did not, as expected, act as shikimic acid precursors, whereas ~ - ( l - C l ~ ) glucose gave shikimic acid of specific activity comparable to that of the precursor. With the use of glucose labeled in various positions, it was found (804, 805 as modified by 805a) that the shikimate carboxyl carbon arises from C-3 and C-4 of glucose; C-1 of shikimate arises from C-2 and C-5 of glucose; and C-2 of shikimate arises from C-1 and C-6 of glucose. Thus the carbon triad carboxyl . . . C-1 . . . C-2 of shikimate is in all probability derived from a three-carbon product of glycolysis. The remaining four-carbon portion of shikimate has a more complex origin, with C-3 to C-6 of shikimate corresponding in some degree to C-3 to C-G of glucose, but with C-1 of glucose also contributing to shikimate C-6 to a significant extent (cf. diagram 5). The results are compatible with the postulated role of sedoheptulose diphosphate as an intermediate in the glucose-shikimate conversion (45Ga,b, 823a).

B a CIIr' CH(XlIz) C02H 6 CHAOH CO,H

Ho-. 5 0 @ f$ <4 3 HO 4. 'OH HO" , "OH OH (OW OH

a-D-Glucose Shikimic acid Phen>lalauir,e and tyrusine Diagram 5 . Numbering of carbon atoms in various substances discussed.

46 C. E. DALGLIESH

The isotope labeling of the benzene ring of tryptophan formed from carboxyl-labeled acetate and (3 ,4-CI4) glucose suggests that this has the same origin as the benzene ring of tyrosine and phenylalanine (686a,b), thus providing strong confirmatory evidence for the common-precursor pathway.

The information at present available is thus sufficient to exclude certain possible routes for aromatic biosynthesis, but is not yet sufficient to reveal the actual mechanism or mechanisms used, or to define parts of the pnth- way. But it seems probable that the techniques available are adequate to deal with the problem and that perhaps in a short time the present obscuri- ties will be made clear.

Further information and pertinent speculations 011 aromatic biosynthesis can be found in reviews by Ehrensvard (212) and Davis (191).

111. DEGRADATION O F PHENYLALANINE AND TYROSINE TO ACETOACETATE ; THE PRINCIPAL ROUTE USED BY nfAMM.4LS

The distinction between ketogenic and glycogenic substances was one of considerable importance to the earlier biochemists, and that phenylalanine and tyrosine are ketogenic was established a t an early date. Embden and co-workers in 1906 (226) showed that phenylalanine and tyrosine yield acetoacetic acid when perfused through a surviving liver. The ketogenicity was confirmed by numerous workers (e.g., 42, 131, 169, 208, 225, 706, 902) using classical methods and was put beyond all doubt by use of isotopically labeled precursors (905, 933). Phenylalanine and tyrosine were later shown also to be glycogenic (130, 131), and this, too, has been confirmed isotopically (552), the glycogen and keto-bodies being derived from different parts of the amino acid molecule.

In this review i t is hoped to give some idea of the historical development of the subject as well as of its present status, and the elucidation of the pathway for the conversion of phenylalanine and tyrosine to acetoacetate is therefore set out in a manner corresponding in some degree to the development of knowledge on the subject.

IIIA. EVIDENCE I~ERIVED FROM IXBOF~N ERROW OF A ~ E T A B O L I S M In discussing the use of mutants of microorganisms in the study of aro-

matic biosynthesis it was pointed out that valuable information could thus be obtained. An organism with a metabolic block rendering i t unable to convert a substance X into its metabolite Y is likely either to excrete X, or to metabolize X by an alternative pathway if such is available, or to excrete metabolites of X formed by the action of relatively unspecific “detoxicating” systems. Accumulation or excretion of abnormal substances may therefore indicate an erizyniic deficiency of this type. In the latter part of

METABOLISM O F T H E AROMATIC AMINO ACIDS 47

the last century there came to be recognized various rare humaii disorders in which excretion of abnormal substances occurred. The reinarkable in- sight of Garrod (ass), in particular, led to the realization that these “inborn errors of metabolism” were due to a congenital iiiability to perform a normal metabolic reaction; the patients were in fact natural mutants providing invaluable material for metabolic studies. Comparatively few metabolic disorders of this type are known; it so happens that an appreciable proportion of these are related to pheiiylalanine and tyrosine metabolism and gave the first clues as to metabolic pathways. The relationship of these defects is summarized in diagram 6, aiid the evidence for each will be discussed. Three of the defects, alltaptonuria, tyrosinosis, arid phenylketonuria will be considered immediately, and a fourth, albinism, will be considered (p. 69) under an alternative pathway for tyrosine metabolism.

Blocked iri

plienylketoouria

Phenylalanine &* lyrosine - 3,4-DihydroxyphenyIalanine

I t I I t Blocked iu + albinism

Phen ylpyruvic acid p-Hydroxy’I)henylpyruvie acid RIeIanin, I etc.

-t Blocked in tyrosinosis

2,j-Dil,ydroxyplieiiylpyruvic acid

I hom*ogentisic acid + Blocked in

alkaptonuria

Acetoacetic acid, etc.

Diagram 6. Relationship of metabolic defects concerned with phenylalanine and tyrosine metabolism. For structural formulas see diagrams 8, 11, and 12.

i . Alcaptonuria, and Related Work on M a n or Intact An imal s

For centuries rare cases have been noted (cf. 286) of persons passing urine which turned black. The condition was first accurately described in 1859 by Boedeker (86), who found that the urine contained a strongly reducing chromogen which he called “alkapton.” The chromogen was first isolated in 1891 by Wolkow and Baumanii (949) and identified as 2,5-dihydroxyphenylacetic (hom*ogentisic) acid (949, cf. also 638, 657; structure, diagram 8). Wolkow and Baumann showed that hom*ogentisic acid was derived

48 C. E. DALGLIESH

from tyrosine but thought that it was primarily a product of bacterial metabolism. Abderhalden ct al. (1) made a bacterial origin seem less likely by subcutaneously injecting glycyl-L-tyrosine into an alkaptoriuric aiid finding exccss honiogtwtisic acid excretion. Excess hom*ogrntisic acid excretion also occurs on :Idministering phenylalanine (250). hfeycr (597) and Friedmaiiii (279) postulated that the side chain of hom*ogentisic acid arises from migration of the side chain of phenylalanine and tyrosine.

The metabolic defect involved in alkaptoiiuria was suggested by Bateson (34) as early as 1902 to be inherited as a recessive Mendelian character, and later evidence has supported this prediction (395, 643). Gross (322) in 1914 concluded that it was due to lack of a specific enzyme. Alkaptonuria, unlike phenylketonuria, is not accompanied by mental symptoms arid is not an incapacitating disorder except insofar as it may lead to ochronosis and arthritis (cf. 598).

hom*ogentisic acid is excreted by the alkaptonuric 011 giving tyrosine or p-hydroxyphenylpyruvic acid, but not on giving o- or m-hydroxyphenyl- alanines or their corresponding keto acids (84, 636), and is also excreted on giving 2,5-dihydroxyphenylpyruvic acid (636). hom*ogentisic acid gives acetoacetic acid when perfused through a surviving liver (226) and so does p-hydroxyphenylpyruvic acid, but not phenylpyruvic acid (225). It soon became accepted that a probable pathway of phenylalanine metabolism was as follows:

phenylalanine -+ tyrosine -+ p-hydroxyphenylpyruvic acid

I acetoacetic acid +- hom*ogentisic acid

This picture (correct as later evidence has shown) was complicated by a number of other obscrvations. Phenyllactic acid was apparently ketogenic, whereas p-hydroxypheiiyllactic acid was not (226). Phenyllactic acid apparently iiicreased hom*ogentisic acid excretion in the alkaptmuric, whereas p-hydroxyphenyllactic acid did not (636). Thesc ohservatioiis suggested that phenyllactic acid was also on the normal metabolic pathway, but it was not clear how it fitted into the chain. The difficulty was partly resolved in 1935 when Edsoii (208) showed that the ketogenicity of pheriyllactic acid mas due to the use of the ammonium salt, and that it was the ammonia aud riot the phenyllactate which was ketogenic; on the other hand, p-hydroxyphenylpyruvic acid and hom*ogentisic acid were truly ketogenic. A complete and critical account of the large amount of early work on alkaptonuria (up to 1928) has been given by Keubauer (637).

Additional interest arose when it was found possible to produce experimental alkaptonuria in animals. This can, for example, be brought about

METABOLISM OF THE AROMATIC AMINO ACIDS 49

by feedirtg the rat or mouse excess phenylalanirie (130,527,528, 662). Sea- lock and Silberstein (793) then found that in the guinea pig given excess tyrosine the excretion of hom*ogen tisic acid was inversely proportional to the ascorbic acid intake, and that administration of excess ascorbic acid could prevent hom*ogentisic acid excretion. Moreover hom*ogentisic acid excretion occurred on giving tyrosine to the human being on an ascorbic acid-free diet and ceased when ascorbic acid was also administered. The hom*ogentisic acid excretion differed from that found in true alkaptonuria in being accompanied by excretion of considerable amounts of p-hydroxyphenylpyruvic acid and p-hydroxyphenylacetic acid, it being possible in some cases to account for the greater part of administered tyrosine as these three metabolites (794). A similar excretory picture was obtained on giving phenylalanine (792, 794). This work showed, as is discussed in more detail later, that ascorbic acid is connected with tyrosine and hom*ogentisic acid metabolism. However, human alkaptonuria is not due to a simple ascorbic acid deficiency, as additional ascorbic acid does not prevent, hom*o- gentisic acid excretion (199, 610, 643,788).

Experimental alkaptonuria has also been produced in rats on a diet de- ficent in sulfur-containing amino acids (295). Pimilar excretory patterns were produced after additional phenylalanine, tyrosine, or their corrc- sponding keto acids, and thc condition was relieved on giving cysteine, but not ascorbic acid (644). Moreover the p-hydroxypheuylpyruvate excretion was much lower, relative to the hom*ogentisic acid excretion, than in the type of ascorbate-dependent alkaptonuria studied by Pealock in the guirien pig (rats cannot in any case be made ascorbic acid-deficient). Seuberger and Webster (G44) also showed that this second type of experimental alkaptoriuria could be produced in many types of amino acid im- balance, or in protein deficiency, and that the threshold intake of phenylalanine or tyrosine required to produce the condition varied with the nutritional state and also with the acid-base balance, acid urines heing asm- c-i;ttrd with a decreased hom*ogentisic acid excretion (cf. also 160, 273, 787).

Seubauer (637) thought it possible that the initial formation of a 2 , 5 - dihydroxyphciiyl compound took place at the amino acid stage to giw

various routes, e.g. :

C. E. DALOLIESH

2’,6-I)ihydroxyplieiiylpyruvic acid

or hom*ogentisic acid

2,5-Dihydi oxy~~lienyletliylurnlllc

2,5-Dihydroxypheriylalariine was synthesized arid resolved by Keuberger (641) and its metabolism inan alkaptonuric examined as part of a detailed and extensive investigation (643). The conversion of pheriylalanine or tyrositie in an alkaptonuric was estimated to be 70 % t o 90 %. hom*ogrntisic acid was shown to be actively secreted by the kidney tubules, the concentration in blood being very low even after giving pheiiylalanine. 2 , s - Dihydroxyphenylalanine gave rise to hom*ogentisic acid excretion, but the evidence was insufficient to show whether i t was a normal intermediate. T,aiiyar (529) found that in the alkaptonuric phenylpyruvic acid was converted to hom*ogeritisic acid with a high efficiency, whereas the conversion of p-hydroxyphenylpyruvic acid was much lower, arid that in experiment a1 alkaptonuria in rats neither of the keto acids could induce alkaptonuria or maintain an alkaptoiiuria previously induced by tyrosine. He therefor(. coiicluded that the keto acids are not normal intermediates in hom*ogcii- tisic acid formation. I-auyar’s findings are opposed by earlier results (225) and by all the enzymic work on the subject, discussed later, and their expla- riation is not obvious.

Experiments on, or related to, alkaptonuria led t o a provisiorial picture of phenylalaniue and tyrosine metabolism shown in diagram 7 .

Phenylalanine phenylpyruvic acid . phenyllactic acid

V V V

I , I I

I ? I ? I ?

Tyrosinc p-hydroxyphenyl- . p- hydroxyplienyl- I pyruvic acid lactic acid

: ? : 1

v V 2,SDihydroxyphenyl- <:: ::=> 2,5-dihydroxyphenyl-

alanine pyruvic acid I I

V i ? V

2,ii-Diliydroxyplienyl- - -- --* 2,5-dihydroxyplienyl-

Diagram 7 .

- fui ther metabolism phenyletliylamine acetic (hom*ogcntisic) acid

Possible pathways for hom*ogentisic acid formation suggested b y early work, particularly on alkaptonurics.

METABOLISM OF THE AROMATIC AMINO ACIDS 51

This picturc has been clarified t)y cnzyinic experimcnts which will be described later. Various other comments are, however, appropriate here. 2,5-Dihydroxyphenylalanine can be converted to 2,5-dihydroxyphenyl- c%hylamine (83), which is a substrate for amine oxidase (80) and which if formed would therefor(. be expected to give lionlogentisic acid. It is uii- likely that either phenylacetic acid or p-hydroxypheiiylacetic acid could be intermediates in hom*ogentisic acid formation, as these are known to be treated as foreign substances by the body and converted to glycine con- jugates (cf. 930) ; moreover p-hydroxyphenylacetic acid does not give hom*o- grntisic avid in the alkaptonuric (635). The formation of a 2,5-dihydroxyphenyl compound must therefore be brought about a t the amino acid or correbponding keto acid stage, or, much less likely, from the lactic acid derivative. . 2. Tyrosinosis and Other Cases of p-Hydroxgphenylpgruvic Acid Excretion

In 1932 RiIedes (590) reported on a patient who excreted large quantities (c. 1 .G g. per day on a normal diet) of p-hydroxyphenylpyruvic acid. This condition she named tyrosinosis. On adding increasing amounts of tyrosine (either pure or as protein) to the diet, the p-hydroxyphenylpyruvste excretion increased and further excretory products appeared in the order, tyrosine, p-hydroxyphenyl-L-lactic acid, and 3,4-dihydroxyphenyl-~-alanine. Additional phenylalanine gave rise to increased excretion of p- hydroxyphenylpyruvate and also of tyrosine and p-hydroxyphenyllactate ; additional p-hydroxyphenylpyruvate was excreted mostly unchanged and partly as p-hydroxyphenyllactate, but no tyrosine was excreted; additional p-hydroxyphenyllactate was excreted unchanged; hom*ogentisic acid was metabolized completely. Medes concluded (1 ) that p-hydroxyphenylpyruvate formation was an early step in tyrosine metabolism, (2) that p - hydroxypheiiyllactate was the product of a side reaction, formed enzymically as shown by the optical activity, (3) that in tyrosinosis the formation of 2,5-dihydroxyphenyl compounds is prevented, tyrosinosis involving a block one stage earlier in the metabolic chain than that involved in alkaptonuria.

It is fortunate that Medes was available to make such a complete and able investigation, as no further cases of tyrosinosis have been reported and the metabolic defect must therefore be exceedingly rare. (There having been oiily one case recorded iiothing is, of course, known of the genetics of its inheritance). Recently, however, other cases of p-hydroxyphenylpyruvic acid excretion have been observed, and though these are probably of a different type it is convenient t o consider them here.

Felix and co-workers developed a specific method for estimating urinary p-hydroxyphenylpyruvate (549) and then investigated the possible use of

32 C. E. DALGLIESH

this substancr for testing liver function (258, 260). After giving p-by- (IroxyDhciiylpyruvate to norma! persons arid patients with liwr dgsfunctioir the intensity of the. Millon reactioii of the urinc. w:ts roughly proportional i o the dcgrce of liver d:image. Much of the Nilloii rcaction was chiti to phe.rtol arid not to unc~haiigetl p-hydroxyphcnylpyrii~ at c, a i d quite apart from i hr

ibility of thc starting material thc method tloes itot, make ;t good liver function test ( c j . 313). During this work, however, two cases were eiicountered in which spontaneous p-hydroxyphenylpyruvate excretion occurred (259, 546). Gros and Kirnberger (313) then made an extensive survey and found p-hydroxyphenylpyruvate excretion to be much less rare than was previously supposed. It was observed in 99 of 122 cases with liver disease (iiicluding all of 43 cases with liver cirrhosis and none of 4 cases with fatty liver) and in 35 out of 41 cases with miscellaneous blood disorders (including all cases with severe anemias). The amount excreted was far less than in tyrosinosis; in liver disorders excretion values went up. to 100 mg. per day with a mean of 40 mg. per day, and in blood disorders excretion was rather less. Moreover exogenous tyrosine seemed not to be involved, no increase in p-hydroxyphenylpyruvate excretion being observed even after considerable additions of tyrosine to the diet. The excretion was attributed to a decrease hi the general oxidative capacity of the liver, due cither to cellular damage or an inadequate oxygen supply. p-Hydroxy- phenylpyruvate excretion was also observed in various miscellaneous disorders such as endocarditis and Salmonella infections.

p-Hydroxyphenylpyruvate excretion can also occur in aiiiinals, for example, in the rabbit fed large amounts of phenylalariirie (511) and, as already noted, in experimental alkaptonuria ( e g , 295, 644). Both p-hydroxyphenylpyruvate and 2,5-dihydroxyphenylpyruvate are excreted on giving sodium butyrate aiid tyrosine to the guiiiea pig (852). When given in large amounts to ftnimals p-hydoxypheriylpyruvate is in part converted to p-hydroxypheiiylacetate (471) ; the same conversion can occur in man.

Levine and co-workers (555-557) made the important observation that p-hydroxyphenylpyruvate (and also p-hydroxyphenyllactate, but not in general hom*ogentisic acid) is excreted by prematuie infants especially when the intake of phenylalanine and tyrosine is high. This excretion ceases on giving ascorbic acid, suggesting that asrorbate is concerned in the further metaholism of p-hydroxypheiiylpyruvate (cf. also 961). This coriclusioii was strengthened by the finding, already discussed, that, p-hytlroxyphenyl- pyruvate is excretrd i n artificial scurvy in animals aiid man (e.g., 792, 794).

3. Ph~nyllictonrrria (Oligophrmia Phenylpyni i~ ica , Folling’s Syndrome)

Folling (272) in 1934 first described a syndrome characterized clinically by mental defect and hiochemically by the presence in the urine of phenyl-

METABOLISM OF THE AROMATIC AMINO ACIDS 53

pyruvic acid (cf. also 445, 44G). The disorder is a good deal more widespread than alkaptonuria, the number of cases in Great Britain being estimated at 1600, or about 4 per 100,000 population (615), but there are considerable geographical differences in its incidence, which may be due partly to inbreeding in isolated commuiiities and partly to the ultimate racial group. It has been calculated (cf. 138) that about 1 in 200 people carry the recessive gene of phenylketonurin. There is some evidence that carriers are more liable than noncarriers to mental instability (663a). Fortunately at present few, if any, pheiiylketonurics have offspring, but if treatment were sufficiently improved to allow phetiylketouurics to reproduce, thc incidence of the disorder might he expected to rise.

The urine also contains considerable amounts of phenylalanine (180, 274), phenyllactie acid (150, 151), atid phenylacetylglutamine (832, 960), typical urinary excretioii values being (9GO), in milligrams per 100 ml., phenylalanine 38, phenylpyruvic acid 50, phenyllactic acid 54, and phe- nylacetylglutaminc 53. Pheriylacetylglutamiiie oc('urs in iiormal urine in appreciable quantities (0.25 t o 0.5 g. per day), but excretion by thc phenylketonuric is much higher (e.g., 2.4 g. per day, ref. 832).

Folling suggested that the fundamental metabolic error was an abnormal racemization of phenylalaninc, thc D-isomer giving rise to the phenylpyruvic acid. 13ut although there are ahnormally high amouiits of phenyl- :danine in the blood of the phenylketonuric (274, 450), iioiie of this is of the wconfiguration. Penrose and Quastel (664) suggested that the metabolic error was aii iiiability to break down pheriylpyruvic acid. But the blood of these patients contains negligible phenylpyruvic acid, and both phenylpyruvic aiid phenyllactic acids can be aminated by the phenylketonuric (450). (Subsequently Jervis (448) has found small amounts (c. 0.7 mg. per milli- liter) of phenylpyruvic acid in the blood of phenylketonurics, this being increased on administering phenylalaninc, wpecially the D-form, or phenylpyruvate). The correct interpretation wits made in 1947 by .Jervis (447), who showed that in the normal fasting animal or man ingestion of phcnyl- alanine or phenylpyruvic acid is followed by a rise in Millon-reacting (i.e., phenolic) substances in the blood, whereas in pheny1ketonuric.s this docs not occur. He therefore deduced that the metabolic error in phenylketonuria is an inability to bring about hydroxylation. It was still uncertain whether the substance normally hydroxylated is phenylalanine (giving tyrosine directly) or phenylpyruvic acid (the conversion of phenylalanine to tyrosine therefore occurring via the keto acids), or whether both types of hydroxylation occur (c j . diagram 7).

The direct conversion of phenylalanine to tyrosine was suggested by the carly work on alkaptonuria already described. Direct conversion was

The condition is iiilierited as a recessive Meudeliaii character.

54 C. E. DALGLIESH

rnade very probable by the work of Moss and Schoenheinier (614) iii 1940, who found that in the rat deuterium-labeled pheriylalariine was converted to tyrosine arid that the Conversion continues even iii preseircie of excess tyrosine, so that the process must be automatic and iiidepcirdent of the animal’s tyrosine rcqirenicint. Similarly pheiiyllactic :wid is uutomnt 1-

cally converted to pheiiylalaninc and tyrosine (613). Thcl cwzyiiie coil- veriing phooylalariine to tyrosiiie was isolated from rat liver by lidc~iifricmcl a i d Coopcr (886) and partially purified (the dctails of this reaction arts (lib- cussed later). When Jervis (149) examined livers of pheiiy1ketoiiui.ic.s souir after death using Utlcnfriend arid Cooper’s conditioiis hc could find no evidence for any formation of tyrosine froin phenylalaninc, whereas livers of controls showed a high conversion. The metabolic defect in phenylkc- tonuria was thus finally tied dowii :IS a failure to hydroxylate phenylnlanirrc to tyrosine.

Afore recent isotopic investigations by Uiidenfricnd and Beseinaii (880) have shown that a small conversion of phenylnlanine to tyrosine can occur in phenylketonuria. Four possible explaiiations of the primary block werc advanced: (1) there may be a reduced amount of the appropriate enzyrne system, L-phenylalaiiitie oxidase, in the livcp, or (2) ti complete absence of the enzynic with conversion by alternative pathways (cf. 174), or ( 3 ) a iior- ma1 amount of apoeiizynie with a coenzyme inissing or (4) a normal amount of enzyme inactivated by an inhibitor. It is known from work on microorganisms that metabolic blocks are by no means always complete (cf . , e.g., 187, 188) and this may well apply to phenylketonuria, but which on(’ or more of the above four possibilities is correct must await further work.

The relation cif tht. biochemical findings to the mental symptoms in phenylketoiiuria is still obscure. Toxicity of abnormal nietaholites actiiig over long periods is an obvious possibility. The finding (17b, 64, 65,961a) that the mental conditioii improves on a continuous low-phenylalaniiie diet, provided this is started before pernianent brain damage appears, gives some support to this cwiicept. It is to be expected that a deficiency of tyrosine, to which the phenylketonuric is liable, may also contribute t o the symptoms. The abnormal response of the phenylketonuric to adrenaline (138) may be due to a lowered endogenous adrenaline production due to a lack of tyrosine. The excretion of abnormal indolic metabolites (17) may indicate that tryptophan metabolism is also modified in phenyl- ke t onuria .

The absence of pigmentation associated with phenylketonuria may be due either to lack of tyrosine as substrate for melanin formation, or t o inhibition of tyrosinttse by the abnormal amounts of phenylalanine (179a), or to both. Pigmentation is restored on giving tyrosine (816a).

The cxisttwce in phrnylkrtonuria of a single metabolic block is in accord

METABOLISM OF T H E AROMATIC AMINO ACIDS 55

with the apparent determination of inheritance by a single recessive gene. Ho\vevcr, Boscott and Bickel (97, 98, and cf. 16) have found that the urine of phenylketonurics contains metabolites such as o- and p-hydroxyphenylacetic acids and p-hydroxyphenyl lactic acid (cf. 17a, 17c). They explained these by postulating that ortho-hydroxylation played a part in normal phenylalanine metabolism and that in phenylketonuria there were a whole series of metabolic blocks. Dalgliesh (174) has shown that the hydroxylated metabolites are probably due to action on the abnormally accumulated nonhydroxylated metabolites of the normal detoxicating mechanisms of the body, and that a single metabolic block adequately explains the known facts.

IIIB. ENZYMIC EXPERIMENTS ON THE NORMAL PATHWAY IN MAMMALS

The work already described on man and intact animals allowed a tentative scheme to be formulated for phenylalanine and tyrosine metabolism (diagram 7). In this section the enzymic and isotopic experiments will be described which have filled in many details of the picture. The subject will again be outlined in a semihistorical manner, but for convenience the complete pathway is summarized at this stage, in diagram 8.

1. General Outline of the Pathway

Early experiments by Bernheim, Felix, Sealock, and their co-workers on oxidation of tyrosine by liver breis showed an uptake of four atoms of oxygen per mole of tyrosine, with the production of one molecule each of carbon dioxide and acetoacetate, but no ammonia (60, 61, 261, 262, 789, 976). Felix and Zorn (261) found alanine to be formed and considered this to arise from a direct splitting of the tyrosine side chain. Although the experiments with man and intact animals already described made it seem very probable that p-hydroxyphenylpyruvic acid and hom*ogentisic acid were normal intermediates in tyrosine metabolism, and although ho- mogentisic acid was known to be readily metabolized by normal liver (e.g., 208, 695, 976) Felix and co-workers (262) considered p-hydroxyphenylpyruvic acid and hom*ogentisic acid not to be intermediates in the breakdown of tyrosine by the liver system.

Isotopic experiments shed more light on the contradictory evidence. Weinhouse and Millington (905) incubated liver slices with tyrosine labeled with C14 in the 0-position of the side chain, and found that the activity appeared in the methylene carbon of acetoacetate. Schepartz and Gurin (772, 773) incubated with liver slices phenylalanine labeled with C14 in the carboxyl group or a-position, or in positions 1 , 3, and 5 of the aromatic ring. They found that the a-carbon of the side chain became the carboxyl-carbon of acetoacetate; either C-1 or C-3 of the ring became the terminal methyl

56 C. E. DALGLIESII

Id -phenylnlnninc transnminntion,

e.g., xitli

Phenylalaniric Tyrosine

(cofnctors: nscorbic wid,

6ce disgrnnis 9 and 10)

$C&*CO -CO,H H O O C H , . C O . C O J I - ? vitamin B,z.

HO p-HgdroxyphenJ.lpyruvic acid 2,5-Dihydroxyphenj Ipyruvic acid

1 direct conversion

lioningent isicnse (cofactors: ferrous iron,

CHzCO,H &C"2*Co2H 1 ascorbic 1 iolie acid) acid, * CO. CHt

HO' AIaleylacetoacetic acid hom*ogentisic acid

+ CH,*CO*CHz*CO,H CO. CHz I

Furnarylscetoacetic acid Fumaric acid Acetoacetic acid Diagram 8. Summary of the steps involved in the degradation of phenylalanine

and tyrosine to fumaric and acetoacetic acids by the principal pathway used by m i - mals and man.

of acetoacetate (thus providing a direct proof of the suggestion of Meyer (597) and Friedmann (279) that the phenylalanine side chain shifts to an adjacent ring carboiil ; there was no randomization between methyl and methylerie carbons of acetoacetate, so that the latter must have been derived from an intact four-carbon unit, and no participation of two-carboil units occurred in the degradation. Lerner (552) synthesized phenylalanine labeled uniformly in the ring with C1* and labeled in the a-position of the side chain with C13, and incubated this with liver slices. The dilutions of C14 and C13 in the acetoacetate mere the same, showing that breakdown and resynthesis could not have occurred. Lerner considered the fate of the four ring carbon atoms not accounted for, and concluded that i t was unlikely that breakdown to two-carbon units had occurred) so that a four-carbon unit such as malate or fumarate (Neubauer (637) had earlier suggested

METABOLISM OF THE AROMATIC AMINO ACIDS 5 7

formation of the latter) should be formed. He diluted his preparations ivith inactive malic acid which he then reisolated and found to be active. The activity was moreover equivalent in all carbons, consistent with its der- iv:ttion from the original aromatic ring of phenylalanine, and the amounts of acetoacctate and malate formed appeared to he equivalent. Later isotopic work (eg., 201, 752, 933) has supported various aspects of their results, which are all consistent with degradation in the following manner:

ICO~H CH, I I

xCHOH xCO - I + I

(OH) 0 B

Q): . \* - Q '' CH2-?O-H = CH1 I CH1 I

CHz xC02H *CO,H 1

I CO,H

CH*XHz a = ling carbon

= a-carbon of side chain

The apparent conflict between enzymic and isotopic evidence was resolved by the work of La Du and Greenberg (523), Sealock and Goodland (789,790), and, in particular, Knox and Le May-Knox (489,543). a-Keto- glutarate and ascorbate (cf. 661, 700) were found to be cofactors for the tyrosine-oxidizing system of liver (543), and the dialyzed soluble fraction of liver had high tyrosine-oxidizing activity on addition of these substances. As the complete system took up four atoms of oxygen per mole of tyrosine and not the five atoms which would have been required had oxidative deamination occurred, it was concluded (489,523,769) that trarisamination is the first step in tyrosine metabolism; this step can be made rate-limiting, in which case i t shows a dependence on pyridoxal phosphate (489). Trans- amination was confirmed by demonstration of the formation of glutamate from a-ketoglutarate and by isolation of p-hydroxyphenylpyruvate as its 2,4-dinitrophenylhydrazone (489). The system could also use oxalacetate (523) or pyruvate in presence of a-ketoglurarate (489), in which case the corresponding amino acids were formed. 2,5-DihydroxyphenyIalanine was not attacked by the tyrosine-metabolizing system (489) and could therefore be excluded as a normal intermediate (cf. p. 50).

The oxidation of p-hydroxyphenylpyruvate also involved four atoms of oxygen per mole, and ascorbic acid was involved in the first oxidative step (790). Its effect was catalytic and it could be replaced by iso-ascorbic acid but not by substances such as glutathione (489). The oxidizing system was highly specific, oxidizing only L-tyrosine, p-hydroxyphenylpyruvate, 2,5- dihydroxyphenylpyruvate, and hom*ogentisate. Ravdin and Crandall (695) had previously shown that the intermediate formed from hom*ogentisic acid is fumarylacetoacetic acid and the combined results all fitted in with

the following scheme (for structural formulas see diagram 8) :

mate 1. Tyrosine + a-kctoglutarate --+ p-hydroxyphenylpyruvatc + glutti-

2 . p-Hydroxyphenylpyruvate "pt","o"p 2,5-dihydroxyphenylpyruvnt r

3. 2,5-Dihydroxyphenylpyruvate 'lP:agk: F COZ + 1iomogciitib:tttI 4. hom*ogentisate o2 F fuinarylacetoacetate 5 . Fumarylacetoacetate 3 fumarate + acetoacetate This scheme has been confirmed in numerous experiments, especially

those of Felix and co-workers (257) in which they re-examined and clarified their earlier results.

The various steps, including some which have more recently become cvi- dent, will now in turn be considered in detail. Before doing this, however, comment should be made on the site of the normal degradation of tyrosine. All the above work was carried out on liver slices, hom*ogenates, or extracats derived from many different species, and the liver probably makes the pre- dominant contribution to tyrosine degradation. Degradation can, however, occur in other organs. The kidney can also carry out tyrosine degradation by the same pathway as in liver but more slowly (160, 101), oxidation in kidney, like liver, hom*ogenates depending on availability of keto acid (164). Tyrosine oxidation by kidney extracts can also lead to accumulation of p-hydroxyphenylacetic acid (257, 724), possibly owing to loss of ascorbate in preparation of the extracts. Brain, intestine, spleen, and blood appear to be unahle to oxidize tyrosine (160); indirect evidence has suggested that some oxidation may occur in muscle (314), though this is not supported by experiments with hom*ogenates (160).

uptake of

2 . Conversion o,f Phenylalanine to Tyrosine

This transformation, Considered likely on the basis of experimeuts with the intact animal, was proved by the isotopic experiments of Moss and Schoenheimer (614), which also showed it to be an automatic process. Udenfriend and Cooper (886) found a highly specific enzyme in liver mhichh carried out this reaction, possibly in two stages. The system required oxygen and DPN (diphosphopyridinenucleotide) and was considered responsible for the greater part of the normal metabolism of phenylalanine.

Mitoma and Leeper (605) resolved the system into two components, separated from rat liver hom*ogenate supernatant by differential precipita- tion with ammonium sulfate. Their combinedsystem of enzyme I, enzyme 11, DPN (or reduced DPN), and an aldehyde or alcohol, specifically hydroxylated L-phenylalanine but not other aromatic compounds. Enzyme I is labile and only a tenfold purification could he achieved. Enzyme I1 is relatively stable. Many, but not all, aldehydes and alcohols tested rould participate in the reaction.

METABOLISM OF T H E AROMATIC AMINO ACIDS 59

The conversion of phenylalanine to tyrosine occurs in muscle as well as liver extracts (560) and is decreased in the liver of ACTH-treated rats (428). The reverse transformation, of tyrosine to phenylalanine, does not occur even in phenylalanine-deficient animals (309).

3. Conversion of Tyrosine to p-Hydroxyphenylpijruvic Acid The experinicnts described earlier showed that in liver hom*ogenates and

extracts this reaction is brought about by transamination, which is an oblig- atory first step in the oxidation of tyrosine by such systems. The existence of such a transamiiiating system was already known (133, 134, 393), and the observed pyridoxal phosphat e-dependence when transamination mas was made rate-controlling (489) was in accordance with the known behavior of transamiriases (cf. 482).

The absenre of oxidative deaniination was shown by the oxygen uptake and the abseuve of ammonia formation (257, 489, 523, 769). Such results with tissue extracts indicate only that no oxidative deaminase survived the isolation procedure. On the other hand, in rats with a severe pyridoxine deficiency no interference with over-all tyrosine metabolism was observed (141), and though these results are inconclusive because of uncertainty of the degree to which pyridoxine deficiency interferes with transaminase activity, they are supported by the finding (134) that liver of some species, including the rat, contains a powerful tyrosine deaminase (cf. 68, 69). It seems probable that in the intact animal both routes for conversion of tyrosine to p-hydroxyphenylpyruvic acid are used. Slight support derives from results of Schoenheimer and co-workers, who, in the first metabolic experiments with a Ws-labeled amino acid, found tyrosine nitrogen to appear in the amide- as well as amino-nitrogen of other amino acids with a distribution similar to that obtained after giving NI5-labeled ammonia (774).

4. Conversion of p-Hydroxyphenylpyruvic Acid to 2,5-Dihydroxypheny1- pyruvic and hom*ogentisic Acids. Function of Ascorbic

Acid and of Hematopoietic Factors The intermediate formation of 2,5-dihydroxyphenylpyruvic acid in this

conversion has not been proved by isolation. But as this is readily metabolized by tyrosine-oxidizing systems (e.g., 489), unlike the possible alternative intermediate p-hydroxyphenylacetic acid, the pathway is not in doubt. On the other hand, the detailed mechanism of this conversion is probably the major unsolved problem in the study of tyrosine metabolism.

The influence of ascorbic acid on tyrosine metabolism in man and intact animals has been discussed under alkaptonuria and tyrosinosis (q.v.). Ty- rosyluria, also called hydroxyphenyluria, i.e., the excretion of p-hydroxyphenyl compounds in the urine, can be affected by factors other than ascor-

60 C. E. DALGLIESH

bic acid. It can occur, for example, in many types of congenital or acquired anemia in animals and man (e.g., 846) and in steatorrhea (99). A few such cases are relieved by hematopoietic factors, such as folic acid or vitamin Bu as well as by ascorbic acid (182, 648a, 847, 952, 953), though more usually such factors relieve the hematological symptoms without affertiiig the tyrosyluria (588, 589, G11, 747-749). Livers of folic acid-deficient rats have a redured capacity to oxidize tyrosine (722). Vitamin Blz has iio

effect on the excretion of hom*ogentisic acid by the alkaptonuric (271). It is uiicertain from all this evidence whether folic acid and vitamiii B12 arc directly concerned in tyrosine metabolism, for they could well be iiidircrtly concerned either because of the mutual influence of vitamins upoil cadi other (646,963) or by their effect on the general level of nietabolic activity, or by their influence on the availability of, for example, carriers such BY the cytochrome system. The adrenal, for example, possibly exerts an indirect effect on tyrosine metabolism (33, 749). Positive evideiice on the function of the hematopoietic factors should he given by enzymic experiments, but these, as we shall see below, are still contradictory. Tyrosyluria can also occur in vitamin-A deficiency. This is probably due to the accompanying vitamin-C deficiency (596, 806).

The demonstration by Knox that ascorbic acid is a cofactor in tyrosine metabolism has been discussed earlier. Though ascorbic acid is essential both for this purpose and for the prevention of scurvy, the latter is probably not due to any appreciable extent to inadequate tyrosine metabolism (e.g., 723). Further experiments with liver preparations have shown that a number of other substances can replace ascorbic acid, D-isoascorbic acid being as effective as ascorbic acid itself (160, 489). Many other ene-diols are effective in tissue hom*ogenates (522,791) but not necessarily in the intact 5111-

imal, where poor absorption or retention reduces their efficacy (661,975). Substances such as 3-methylascorbic acid (791), which do not contain an ene-diol structure, cannot replace ascorbic acid.

More detailed enzymic studies of the changes involved in the conversion of p-hydroxyphenylpyruvic acid to hom*ogentisic acid have been made by two groups of workers, Williams and Sreenivasan (927-929) in rat liver preparations, and Uchida and co-workers (878) in rabbit liver preparations. Unfortunately the initial results of the two groups differ appreciably, but not all the conclusions are necessarily mutually exclusive.

The American workers studied liver tyrosine oxidase with the aid of 2,6-dichlorophenolindophenol, which destroys reduced ascorbic acid. The inhibition was only partly reversed by ascorbic acid, and glutathione was found also to be involved (928). Under certain conditions the dichlorophenolindophenol could be stimulatory and was thought to replace yet another factor (927). (The work of the Japanese authors discussed below

METABOLISM O F T H E AROI\IATIC AMINO ACIDS ti1

suggests that this could be vitamin Bl2 .) They then studied aged tyrosine oxidase preparations which had lost their activity (920). Activity was restored by adding ascorbic acid, glutathione, and an extract of heated fresh enzyme which supplied, besides pyridoxal phosphate, another, anionic, factor. They found no stimulatory effect 011 adding DPN or flavinade- ninedinucleotide, and a possible stimulation by folic acid which they considered an artefact. They suggested the mechanism shown in diagram 9, which is based on the presumed mode of action of glyceraldehyde-3-phosphate dehydrogenase (686).

:Or HO -C,H,-CHt*CO * CO,H Ar *CHn*CO.CO,H (glutathione)

OH I

1 Ar * CHz. C: * COZH ton

jtI'0 SG

Ar*CHt*CO-SG + C02 4 (unknoun factor)

Oa Ar 'CHz'CozH (ascorbic acid) * HOzC *CH CH.CO.CH2.CO CHz. COzH

+ GSH Ar = 2,5-dihydroxyphenyl

GSH = glutathione Diagram 9. Pathway proposed by Williams and Sreenivasan (929) for formation

and further degradation of hom*ogentisic acid.

Uchida, Suzuki, and Ichihara (878) isolated a soluble enzyme system (thereby possibly excluding mitochondria1 participation) from rabbit liver, and partially purified it. Two enzymes were involved. The first of these converted p-hydroxyphenylpyruvic acid to 2,5-dihydroxyphenylpyruvic acid. If this enzyme was resolved, vitamin C alone did not restore the activity, but vitamin C and vitamin Blz did. The amount of BIZ required was very low, and they suggested that the true enzyme was a B12 derivative, possibly aquocobalamin hydroxide bound to enzyme protein, and that the function of the ascorbic acid was solely to stabilize the reactive form of the coenzyme. This agrees with the work of La Du and Greenberg (524), who considered the role of ascorbic acid to be quite unspecific. Ascorbate increased the rate of tyrosine oxidation in liver preparations but the net con- sumption was zero, and moreover numerous ene-diols were just as effective on a molar basis. La Du and Greenberg considered that ascorbic acid participates in a cyclic oxidation-reduction and happens to be a substance of the correct oxidation-reduction potential either t o participate directly or to protect some other participant.

Uchida et al. excluded the possible function of ascorbic acid as a peroxide source and considered their enzyme to be an oxidase, not a peroxidase. Their second enzyme converted dihydroxyphenylpyruvic acid to hom*o-

C . E. DALGLIESH

p-Hydroxgphenylpyruvic acid

Cofactor vitamin BIZ, Enrymo I stabilized 111 ieactivc

form by ascorbic acid

and DPN Enzyme II Cofactors eocarboxylase

I 2,5-Dihydroxyphenylpyruvic acid

Cofactnr folie acid

I hom*ogentisic acid

1 Further oxidation products

Diagram 10. Summary of v i e w of Uchida, Suzuki, :ind Ichihara (878) on hoinogcri- tisic acid formation and degradation.

gentisic acid. This was apparently a straightforward oxidative decarboxylation, with cocarboxylase and DPN as cofactors. Folic acid was thought to play no part in the formation of hom*ogentisic acid but to be concerned in its further metabolism. They considered that their stages I and I1 might be interconnected and to some extent mutually interdependent. This recalls the coupled reactions of the tryptophan peroxidase-oxidase system (see p. 83), and La DU and Zannoni (525) consider that the p-hydroxyphenylpyruvate oxidase system resembles tryptophan peroxidase- oxidase in many respects. The views of the Japanese workers are summarized in diagram 10 (for structural formulas see diagram 8).

Knox (483) has purified 100-fold an enzyme converting p-hydroxyphenylpyruvate to hom*ogentisate. The system requires either ascorbic acid or dichlorophenolindophenol, and appears to be much more active than systems previously reported. The conversion occurs in one step. Details of the reaction are awaited with interest especially as 2,5-dihydroxyphenylpyruvate appears to be neither ail intermediate nor an inhibitor (209a).

Keu- bauer (637) suggested the following scheme :

The mechanism of the changes under discussion is still obscure.

No hydroxydienone type of int.ermediate in tyrosiiie metabolism has ever been isolated, but analogous chemical changes can be realized (see below). This scheme was expanded by Neuberger (642), who suggested that the phenolic ion in its para-quinonoid resonance form was oxidized with loss of two electrons to give a positively charged carbonium ion; the latter on reaction with a negatively charged hydroxyl would give the hydroxydi-

METABOLISM OF THE AROMATIC AMINO ACIDS 63

enone.

OQ 0 0 0

Q - Q + Q ’ Q HO R R R R

Such a reaction would be expected only in alkaline solution. Moreover, analogous reactions with the resonance forms of the phenoxide ion carrying the negative charge in the ortho position should give 3,4-dihydroxy compounds. The function of an enzyme in this reaction could therefore be to activate the phenolic molecule at physiological pH and to direct subsequent reaction to give para hydroxylat,ion. The reaction has many analogies with that involved in the biogenesis of thyroxine ( q . ~ . ) .

Witkop (946) has discussed chemical analogies to these changes. Reac- tions of the following type can be realized:

benzilic acid rcarrangemcnt I

HO D O H - R 00

Mechanisms such as the above do not involve ortho-quinonoid or catechol- ,4n alternative suggestion (96) involves ortho-dihy- type intermediates.

droxylation (peroxidation) and a pinacol-pinacolone rearrangement :

\ pinncolpina~ulonc reatrangenlent * Rd H \ 26 \ 6-:q OH OH OH I OH

(or a tautomer)

There is 1 1 0 direct evidence for or against either of these schemes; nor is it obvious how (lither ascorbic acid or vitamin I312 would participate in them.

The function of ascorbic acid iii tyrosine metabolism is complicated by the fact that not only does it participate in more than one stage of the normal pathway, but it can also participate in certain nonspecific reactions. It is not kiionw to what extent the mechanisms of these various functions are related. Nonspecific hydroxylatioii of aromatic (.ompounds (22, 23,

64 C. E. DALGLIESH

106, 881, 882), which can also under some circ*mstances play a part in phenylalanine and tyrosine metabolism (174), is carried out by ascorbic acid (or a number of other ene-diol or diketo compounds) and iron in the presence of oxygen. The active oxidizing agent is not ascorbic acid, dehy- droascrobic acid, or peroxide and is considered (882) to be some unknown oxidation product of ascorbic acid. The results recall the enzymic oxidation of reduced pyridine nucleotides, which in all probability (465, 629) involves a one-electron transfer with a still unidentified oxidation product of ascorbic acid which may be a free-radical electron acceptor of the type of monodehydroascorbic acid. On the other hand, hydroxylation appears to be confined to electronegative sites on the aromatic ring (106, 881) Mechanistic studies of the reactions involved are highly desirable.

5 . Conversion 0.f hom*ogentisic Acid to Maleylucetoacetic, Pumarylacetoacetic, Fumaric, and Acetoacetic Acids

Ravdin and Craiidall (695) isolated a protein fraction from rat liver which converted hom*ogentisic acid to a P-keto acid decarboxylated slowly hy aniline citrate a t 38°C. A second enzyme fraction was obtained which converted this keto acid to acetoacetic acid. The P-keto acid was isolated as its silver salt and was found also to be a dicarboxylic acid and a P-diketone, and to give fumaric and acetoacetic acids on hydrolysis. The proposed formulation as fumarylacetoacetic acid (see diagram 8) has since been amply confirmed. Conversion of hom*ogentisic acid to fumarylacetoaretic acid by a liver preparation involves uptake of the expected two atoms of oxygm (e.g., 489, 523).

In the previous section (cf. diagrams 9 and 10) conclusions were cited that ascorbic acid and folic acid may participate in the reactions involved in hom*ogentisic acid degradation. Our principal knowledge of the enzymes involved is due to Suda and co-workers. They first studied hom*ogentisic acid degradation in a Pseudomonas species (840), from which they were able to obtain a cell-free enzyme preparation, and then studied the reactioii in rabbit liver (841). In the latter they found a requirement for a dialyz- able cofactor arid for ferrous iron. Ferric iron was ineffective and thv function of ascorbate in the reaction was considered to he the reduction of ferric iron to ferrous. In livers of scorbutic guinea pigs they found (842) the level of hom*ogentisicase to be much lower than in riornial animals. h r - tivity was restored by ferrous iron or, less effcrtively, by ascorhic. avid. ad-Dipyridyl (which coordinates with iron) inhibited hom*ogentisicasc :iv-

tion and administration of tyrosine to dipyridyl-treated rats was followcd 113’ hom*ogentisic arid excretion. The influence of iron and ascorbic acid a11cl the irihibitioii by dipyridyl have been confirmed by Crandall (160) mid Schepartz (770, 771).

METABOLISM OF THE AROMATIC AMINO ACIDS 65

The quinonoid form of hom*ogentisic acid, i.e., benzoquinone-acetic acid, is probably not an intermediate in the reaction (160,770, 841), in which it acts as an inhibitor, but may be slowly oxidized. The high redox potential of this quinone makes it unlikely to exist in the free state for any length of time in body fluids. Many other chelating substances, e.g., versene and oxine, inhibit hom*ogentisicase (770,771), and the inhibition is reversed by ferrous iron. The extent of inhibition by versene depends on the time of incubation, indicating that the iron is not simply in ionic association with the apoenzyme. Ascorbic acid can be replaced by many ene-diols (771); this supports a nonspecific action. Glutathione may also be concerned in the reaction, possibly pro- tecting essential -SH groups. An iron-sulfur bond may be involved (163). hom*ogentisicase occurs in kidney as well as liver (160, 161, 164) but not in appreciable amount in other organs.

Direct formation of a fumaric acid derivative (having a trans arrangement about the double bond) from a benzenoid compound (having a cis arrangement about the double bonds) would be surprising. Knox (486) has resolved this difficulty by showing that the more probable maleylacetoacetate is the first product of the reaction, and that this is then converted to fumarylacetoacetate by an isomerizing enzyme. The latter can be separated from hom*ogentisicase by alcohol fractionation and the reaction thereby stopped a t the maleylacetoacetate stage. Maleylacetoacetate is formed from hom*ogentisate with uptake of the expected two atoms of oxygen per molecule. The isomerase requires glutathione as a cofactor (cf. 209, 485).

The enzyme hydrolyzing fumarylacetoacetic acid to fumaric and acetoacetic acids has as yet been little studied. It may be the same as acylpyru- vase (592) or the triacetic acid hydrolyzing enzyme (154).

Iron cannot be replaced by other metals.

IV. TYROSINE DEGRADATION BY THE CATECHOL PATHWAY

3,4-Dihydroxyphenylalanine (for structure see diagram 11) is an amino acid isolated (from the pods and sprouts of Viciu fubu) and first definitely identified in 1913 by Guggenheim (323),who showed it to be identical with synthetic material previously prepared (280). It is widely distributed in certain types of plants (beans, etc.) but is not a normal protein constituent. However, it plays an important part in mammalian metabolism of tyrosine, as it is the precursor of adrenaline (epinephrine), noradrenaline (arterenol, norepinephrine), and melanin. In this review these substances will be considered only insofar as they account for a portion of normal tyrosine metabolism.

60 C. E. DALGLIESH

1. Adrenaline, Noradrenaline, and Their Biogenesis

Adrenaline and noradrenaline (structures; diagram 1 1) are the chemical mediators of sympathetic nervous transmission and as such are indispen- sable for normal function of highcr organisms. Numerous reviews are available on their physiology and pharmacology and on the relation of structure to activity (e.g., 02, 74, 114, 244, 324, 735). Adrenaline was first isolated in the crystalline state from natural sources in 1901 by Takamine (851), and its structure soon proved by synthesis and optical resolution of the racemate to give the Zcwo isomer (168,270,834). Noradrenaline was known as a chemical substance before the remarkably recent realization (cf. 297, 850,875) that it, rather than adrenaline itself, is the chief sympathomimetic agent serving to transmit adrenergic impulses under normal conditions (for reviews see 243, 849).

The assumption of early workers that! adrenaline is derived from phenylalanine and tyrosine was conclusively proved by the demonstration (328, cf. also 888) that phenylalanine labeled in the a-position of the side chain with C14 gave adrenaline labeled in the corresponding position. It is reasonable to assume that tyrosine is an intermediate in this conversion. The further conversion of tyrosine to adrenaline involves four changes: (1) introduction of a further phenolic group, ( 2 ) decarboxylation, (3) introduction of the side chain hydroxyl, and ( 4 ) N-methylation. A great deal about the biosynthesis of adrenaline remains obscure, but i t is nevertheless possible to advance a tentative hypothesis concerning the order in which these changes occur.

The conversion of tyrosine to 3,4-dihydroxyphenylalanine occurs both in vivo in man (590) and in vitro by the action of tissue tyrosinase (205,688). Mammals can decarboxylate both tyrosine (402,407) and dihydroxyphenylalanine (406), tyrosine decarboxylase and dihydroxyphenylalanine (dopa) decarboxylases being quite distinct and separable (405), though both are dependent on pyridoxal phosphate (73, 758, and review 72). In mammals dihydroxyphenylalanine is the most readily decarboxylated of all amino acids, and it is therefore not unreasonable to assume that this is the substrate normally decarboxylated in adrenaline biosynthesis (cf. 74, 75). Support for this concept derives from the fact that both the substrate and the product of the reaction (3 ,4-dihydroxyphenylethylamine; diagram 1 I ) can or do occur in the adrenal (298, 299, 802), and the amine is moreover, like adrenaline and noradrenaline, a normal urinary excretion product (245,404).

If the above are accepted as the first two steps in adrenaline biogeriesis it becomes possible to predict that the remaining two steps occur in the order shown in diagram 11.

METABOLISM OF THE AROMATIC AMINO ACIDS 67

Tyrosine

3,4-Dihydroxyphenylalanine 3,4-Dihydroxyphenylethylamine @+dopa; dopa) (hydrosytryamine; dopamine)

i HO HO

m c H 2 - m . CHI - H o b c H . cH2.NHz 1 -

OH OH Adrenaline

(epinephrine) Noradrenaline

(norepinephrine, arterenol) Diagram 11.

line from tyrosine. The pathway considered most probable for the biogenesis of adrena-

The widespread co-occurrence of noradrenaline arid adrenaline in itself suggests that noradrenaline is the immediate adrenaline precursor. This had been considered probable even before the natural occurrence of noradrenaline was known (70, 71), and the methylation of noradreiialine has since been shown both in vitro in adrenal preparations (110) and in vivo on perfusing the surviving adrenal (1 11). The methyl group can arise from methionine, probably formed from choline, in which the adrenal is extremely rich. A large proportion of the activity of administered (methy1-Cl4) me- thioniiie appears in the adrenal (460, 569).

As three of the four stages in adrenaline biogenesis are thus provisionally established, the remaining stage, introduction of the side-chain hydroxyl, should occur by conversion of dihydroxyphenylethylaniine to noradrenaline. There appears to be little information available on this reaction (cf. 195a).

This picture of adrenaline biogenesis may well be an over simplification. Thus 3,4-dihydroxyphenylserine, but not its N-methyl derivative (177), can also be decarboxylated, to give noradrenaline (63,78); this mould imply that a sequence 3,4 - dihydroxypheriylalanine + 3,4 - dihydroxyphenylserine ---f noradrenaline should be considered. 3,4-Dihydroxyphenylserine can be split to 3,4-dihydroxybenxaldehyde and glycine (594), and this might conceivably be a minor pathway of tyrosine metabolism. In the octopus p-hydroxyphenylethanolamine (octopamine) occurs (373, 762) ; this might imply the existence in this species of one of the sequences tyrosine - tyramine + octopamine ---f noradrenaline, or tyrosine -+ p-hydroxyphenylser-

68 C. E. DALGLIESH

HO T H ~ H - C O ~ H OH NHZ

H O ~ ~ H . C H ~ . N H ~ OH

3,4-Dihydroxyphenylserine Octopamine

ine + octopamine --+ noradreiialiiie. However, iii the mammal, though tyramine can be formed, especially in the pancreas (373), it seems to be cn- tirely metabolized by amine oxidase to give ultimately p-hydroxyphenylacetic acid (762), and such sequences as these would be unlikely to occur.

Many reactions of possible significance in adrenaline biogenesis can also take place nonenaymically, e.g., tyrosine to dihydroxyphenylalanine (13, 18,174, 277,690) ; tyramine to dihydroxyphenylethylamine (106,403,690) ; octopamine to noradrenaline (801). Such reactions might in part be responsible for the contradictory evidence from early experiments with tissue slices and honiogenates (e.g., 198, 247, 777, 778, 896). The site of the biosynthesis of adrenaline is moreover not established, and there is good evidence (888) that the adrenal itself is by no means necessarily the principal tissue concerned. Different steps in the hiosynthesis may occur in different tissues. Different species, or even different organs of the same species, may use different routes for biosynthesis. It has even been suggested (803) that a still unsuspected biosynthetic route might owur.

2. Metabolic Llegradation of Noradrenaline and Adrenaline. Adrenochrome

Adrenaline and noradrenaline can both give rise to melaniris (cf. below). An intermediate in such a transformation of adrenaline is adrenochrome, a molecule stabilized by resonance (359). Adrenochrome has a powerful effect on the maturation of reticulocytes (281) and might play a part in normal physiological processes. The extent to which adrenaline is converted to adrenochrome and melanin, and its significance, is still unknown.

I I CH, CHs

Resonance forms of adrenochrome

The degradation of adrenaline to nonpolymeric materials has been studied especially by Schayer and co-workers, using various isotopically labeled adrenalines (760, 761, 764-766). At physiological concentrations no conjugation (e.g., with sulfate or glucuronic acid) occurs. The molecule is almost entirely degraded by t,he action of amine oxidase, which brings about

METABOLISM O F T H E AROMATIC AMINO ACIDS 69

demethylamination with subsequent excretion of a product still containing both a- and P-carbon atoms of the side chain. (For a review on amine oxidase, see 77.)

3. Melanogenesis and Albinism

Extensive reviews on melanogenesis are available (269, 553, 554, and especially 581), and the subject is dealt with only in outline here (diagram 12).

Tyrosine - tyrosinase H o ~ c ~ ~ H * COlH HO \ +t02,

+ $ 0 2 noneiirymic

- G z G G +

+:Or ""a""'; CH'C02H nonenzymic- internal -

reduction HO \ KH

internal HO oxidntion-

/CH*Co2H reduction, * H

decarboxylation HO

Diagram 12. Pathway for the conversion of tyrosine to 5,6-dihydroxyindole, the precursor of melanin.

The transformations involved in melanogenesis were largely worked out by Raper and his co-workers (205, 682, 688, 689, 691) and have been confirmed spectroscopically by Mason (579, 580). Tyrosine, like many other phenols, is attacked by the copper-containing enzyme tyrosinase (phenol- oxidase; for review see 814) to give dihydroxyphenylalanine. This is further oxidized, enzymically or spontaneously, to the quinone, which by an internal oxidation-reduction cyclizes to 5,6-dihydroxy-2, S-dihydroindole- 2-carboxylic acid; this undergoes spontaneous oxidation to the corresponding quinone (dopachrome (1 13), formerly incorrectly identified with the natural pigment hallachrome) which by a further internal oxidation-reduction, possibly catalyzed by zinc (466), and subsequent loss of COz, gives 5,6-dihydroxyindole, the probable precursor of melanin. The me- lanogen in melanuria is a mixture of conjugated derivatives of 5,6-dihydroxyindole (547, 548, 548a, 563). Similar series of reactions can occur with other derivatives of 3,4-dihydroxyphenylethylamine, such as adrenaline, and these also give rise to melanins.

Polymerization of 5,6-dihydroxyindole probably (1 12) occurs by oxidation to the quinone, which then polymerizes through the 3-, 4-, 7-, and occasionally 2-, positions to give a polymer as in diagram 13.

70 C. E. DALGLIESH

5,B-Dihydroxyindolc (cf. diagram 12)

Indole-5,G-quinone

e.g. b

(*) 8 0

<*I

0 * *

- etc.

Diagram 13. suggested type of polymerization in melanin formation (rj”. Du’T,ock Asterisks represent positions taking part i n polymerisa- and Harley-Mason; 112).

tion reactions, those in hrackets heing less likely.

Alternatively (108) a seven-membered ring might be formed before polymerization as in diagram 14.

polymers 2 Molecules of indole-5,6-quinone

1 i H O n < f i o I

I-I 0 H HO

(or isomers) Diagram 14. An alternative suggested route (108) for melanin formation.

Melanin is an extremely inhom*ogeneous substance, bound to protein, (cf. 581a), and also binding metals such as iron (e.g., 531, 532) . It is probable tthat a “pure” melanin, in the sense of a product derived froni a single precursor, rarely occurs naturally.

Car- cinomatous growths in which abnormal melanin formation occurs are known as melanomas. A congenital metabolic defect in which skin pigmentation does not occur is known as albinism, and is inherited as a recessive Mende- lian character (cf. 40). Albinos occur in many species besides man (e.g., the pink-eyed white rabbit). As adrenalhe formation is apparently un- impaired in albinos, the metabolic block presumably lies in the conversion of dihydroxyphenylalanine to melanin, as shown in diagram A, rather thaii in the conversion of tyrosine to dihydroxyphenylalanine. However, the exact nature of the block has not been established.

It seems probable that melanin is not further metabolized, a t least in

Melanin is the normal pigment of the skin arid mammalian hair.

METABOLISM O F THE AROMATIC AMINO ACIDS 71

the mammal, and can therefore be looked on as a true end product of tyrosine metabolism.

4. The Catechol Pathway in the Insect

The insect cuticle is formed from a water-soluble protein and an ortho- dihydric phenol (677) shown to be 3,4-dihydroxybenzoic acid (679). This phenol probably ‘‘tans” the protein by oxidation to the quinone and reaction with protein side chains, cross-linking the chains and causing a hardening and darkening. Besides 3,4-dihydroxybenzoic acid the cuticle also contains 3,4-dihydroxyphenylacetic acid, 3,4dihydroxyphenyllactic acid, and 3,4-dihydroxyphenylalanine (332, 680, and cf. 331), obviously suggesting biogenesis from the latter. 3-Hydroxykynurenine (q.v. ) may also be concerned in both hardening (678) and darkening (431) of insect cuticles.

V. TYROSINE METABOLISM VIA THYROID HORMONES AND OTHER HALOGENATED DERIVATIVES

In higher organisms a portion of the tyrosine is metabolized via the thyroid hormones. Physiologically these are highly active substances, and the amount of tyrosine metabolized by this pathway is probably relatively small. However, no quantitative figures appear to be available.

The exact nature of the thyroid hormone, whether protein or otherwise, and the physiological and endocrinological aspects, such as the mutual interaction of thyroid and pituitary, cannot be discussed here. Excellent reviews are available elsewhere (e.g., 8, 31, 358, 673, 703, 751).

1. Thyroxine and Its Biogenesis

Baumann (37, 39) was first to show that the active principle of the thyroid gland contains iodine. It was only 20 years later, in 1915, that Kendall (461) reported his isolation, after a most laborious separation, of the pure substance, to which he gave the name thyroxine (462, 463). The analytical difficulties with a substance containing some 65 % of iodine are considerable, and the analyses of Kendall’s material misled him into formu- lating it as an iiidole derivative. Harington (349) considerably improved the isolation procedure and showed that Kendall’s formulation was unten- able. Harington also found (350) that iodine c+ould be removed from thyroxine by catalytic hydrogenation to give an iodine-free phenolic amino acid, desiodothyroxine, which was latcr named thyronine.

Degradative studies suggested the presence of two benzene rings, a phenolic oxygen, an ether oxygen, and an alanine side chain. Of the possible structures, that nou7 accepted for thyronine was considered most likely and was proved by synthesis (350). The position of the iodine

72 C. E. DALGLIESH

Thyroxine, R = I Thyronine, R = H

NH1 R R

atoms in the previously known iodo-gorgoic acid (3 , 5-diiodotyrosine; described later) suggested that in thyroxine the iodine occupied the 3,5,- 3' , 5'-positions. As direct iodination of thyronine introduced only two iodine atoms, a new synthesis was devised by Harington and Barger (355)) who prepared 3 , 5-diiodo-~~-thyronine and iodinated this to 3,5,3' , 5'- tetraiodothyronine, identical with the thyroxine from thyroid tissue (this had been obtained by a procedure involving alkaline hydrolysis and conse- quent racemization). The synthetic material was resolved by Harington (351) and the L-isomer shown to be identical with natural thyroxine isolated using nonracemizing enzymic procedures (1 37, 358).

Inspection of the structure of thyroxine suggested tyrosine and diiodotyrosine as precursors (355)) and this supposition was strengthened by the isolation of 3,5-diiodotyrosine from thyroid tissue (276, 357). Between them thyroxine and diiodotyrosine account for the greater part of the organically bound iodine of thyroid. In addition, however, there also occur monoiodotyrosine (265, 860) and triiodothyronine, which is discussed in more detail below. Von Mutzenbecher (566) showed that iodination of casein under appropriate conditions gave a product from which thyroxine could be isolated; moreover, thyroxine was formed to a small extent even on incubating diiodotyrosine in alkaline solution (625). These results were confirmed by Harington and Pitt-Rivers (356), and it was subsequently found (670, 671) that if simple peptide derivatives of diiodotyrosine were used, thyroxine derivatives could be obtained in quite high yields a t pH values nearer neutrality; for example, incubation of N-acetyl-3 ,5-diiodo-~~-tyrosylglut&Illic acid a t pH 7.5 gave N-acetyl-DL- thyroxylglutamic acid in a yield of 36 %.

The study of the thyroid hormones was greatly facilitated when radioactive isotopes of iodine became available. Using II3l i t was soon confirmed (576, 612, 665, 666) that activity administered as iodide was converted in the thyroid to diiodotyrosine and then thyroxine.

The mechanism of thyroxine biogenesis was considered by Johnson arid Tewkesbury (451)) who recognized the similarity between the conversion of diiodotyrosine to thyroxine and the type of oxidation of ortho- and para-substituted phenols extensively studied by Pummerer (684). They suggested that oxidation gave rise to two types of free radical, one with the electron lost from the para position of the quinonoid form and one from the phenolic oxygen. Addition of these two, as in diagram 15, would give

METABOLISM O F THE AROMATIC AMINO ACIDS

H u Q- c y x a m NHZ I

oxidation

1 1

-I- -0 ‘ CHZ.CH(SHZ)*COZH D o@Hza‘:“:’”.:

I I (free radical intermediates)

o ~ ~ ~ * C O ~ H I .1

CHz.CH(NHz) *COzH

I’ I J.

Thyroxine -k amino-acrylic acid

pyruvic acid + h’H, Diagram 15. Scheme of Johnson and Tewkesbury for thyroxin biogenesis.

an intermediate from which they postulated loss of aminoacrylic acid which would be expected to be spontaneously hydrolyzed to pyruvic acid and ammonia. They claimed to be able to detect the latter two substances in their reaction solution, but other workers have not been able to repeat this. Though the main outline of Johnson and Tewkesbury’s hypothesis is accepted, the mechanistic details and the fate of the ejected alanine side chain are not known. The hypothesis was considerably extended by Harington (353) and Neuberger (642), who pointed out that the chemical reaction is likely to occur in alkaline solution and is more probable with diiodotyrosine than with tyrosine itself, as the phenolic group of the former is more acidic. Moreover, the iodine ortho substituents inhibit various other reactions occuring with phenols not so substituted. The only probable product of the reaction is in fact thyroxine. The considerable similarity between thyroxine forinatiori and hom*ogeritisic acid formatioil was pointed out by Neuberger (642), who also advanced an alternative to

74 C. E. DALGLlESH

the free-radical mechanism. Instead of loss of one electron each from two molecules, oxidation of a diiodophenoxide ion could give a positively charged carbonium ion which would then add to a second negatively charged unoxidized diiodophenoxide ion :

+ I’

* Hydroxydienone intermediate

It is not known whether or not the conversion of diiodotyrosiiie to thyroxine is enzymic (cf. 354, 672). If enzymic, the function of the enzyme might be to produce a t a physiological pH the type of reactive intermediate only obtainable in vitro a t a more alkaline pH.

It may be significant that tyrosine, di-iodotyrosine and thyroxine all occur as N-terminal groups in pork thyroglobulin (7204.

More detailed discussions of many aspects of the chemistry and biochemistry of thyroxine are available elsewhere (320, 352, 353, 048, 719).

2 . Yriiodothyronine and Its Riogensis

3,s ,3’-Triiodo-~-thyronine was found in plasma and thyroid by Gross and Pitt-Rivers (316, 318) and identified with synthetic material. It was identified almost simultaneously both free and in the circulating hormone thyroglobulin by Itoche and co-worker,. (714, 715, 717). It is several (probably three to five) times as active physiologically as is thyroxine in many different types of test (317, 319), and the question has been argued a t some length as to which is the true thyroid hormone. The question is not settled, and for the present it is convenient to regard both as hormones or hormone precursors.

There are three plausible pathways for the biosyrithesis of triiodothyronine. It could be formed from one molecule each of mono- and diiodotyrosiiie in the same way as thyroxine can be formed from two molecules of diiodotyrosine; or it could be formed by incomplete iodinatiori of pre- formed thryoiiine or diiodothyronine; or it could be formed by deiodination of thyroxine. Formation by deiodination is favored by English work-

lIo&p,,. . CH I . C02H NHz I

Triiodotliyronine

METABOLISM OF T H E AROMATIC AMINO ACIDS 75

ers (e.g., 320, 570,571) and formation by one of the other routes, by French workers (e.g., 716, 718, 720). Only contradictory evidence is available on enzymes which might specifically bring about synthesis by any of the likely routw. The effect of various thyroxine antagonists has been attributed to inhibition of deiodinatioii (e.g., 923 for references). The biogenetic pathway is still an open question (321), and the coexistence of two pathways is not excluded.

Significant recent papers are 624a, 860a, 863a, 963a.

3. Metabolic Fate of the Thyroid Hormones

Both thyroxine and triiodothyronine are excreted unchanged and as their glucuronides in the bile (476, 721, 859). The greater part, however, probably undergoes deiodination, possibly in the salivary glands (252). An enzyme, tyrosine iodinase, occurs both in thyroid and, to a greater extent, in the salivary glands. This can either synthesize moiioiodoty- rosine from tyrosine, or deiodinate monoiodotyrosine, the direction of reaction varying with the oxidation-reduction potential of the tissue (251, 253). The relation of this enzyme to thyroxine metabolism is still uncertain. It has been suggested that this or a similar enzyme brings about synthesis in the thyroid, where there is a high local concentration of iodine and the product is removed as thyroglobulin, whereas in the salivary gland conditions favor breakdown.

It is not yet known whether deiodination of thyroxine and triiodothyronine is complete or partial, or what route the degradation of the deiodi- nated products takes. Mono- and diiodotyrosine are said not to leave the thyroid as such, but are deiodiriated and the iodine reutilized (320, 720). However, in liver and kidney diiodotyrosine can be converted to the corresponding pyruvic and lactic acids (873), and triiodothyronine and thyroxine give the corresponding pyruvic acids (721a).

4, Other Naturally Occurring Halogenated Tyrosines

Drechsel (203) in 1896 reported isolation from the coral Gorgonia cauo- linii of an iodine-containing amino arid, which he named iodogorgoic acid. Its formulation as 3,5-diiodotyrosine mas established by synthesis (388, 910). It has since been found that halogenated tyrosines are widely distributed in marine organisms, especially in the corneous skeleton of various Anthozoa and sponges. Besides diiodotyrosine there occur monoiodotyrosine, thyroxine, monobromotyrosine, and dibromotyrosine, and their distribution can be used for biological classification. Such organisms concentrate considerable amounts of halogens from sea water, the amount of halogen fixed as halogenated tyrosine depending on the organism’s tyrosine rontent. The field has been well reviewed by Rorhe (713).

76 C. E. DALGLIESH

VI. PATHWAYS OF PHENYLALANINE AND TYROSINE METABOLISM UTILIZED PRINCIPALLY BY MICROORGANISMS

It is probable that the route for pheriylalanine and tyrosine degradation via hom*ogentisic acid, the principal route obtaining in mammals, is also used by at least some microorganisms (e.g., 453, 840). But not all microorganisms necessarily use it, and many can carry out a number of other types of degradative reaction. The chemical potentialities of microorganisms are so enormous that only some of the more important reactions can be considered here.

Baumann (36, 38) suggested the following scheme for tyrosine degradation by the bacterial flora of the gut:

These are summarized in diagram 16.

tyrosine -+ p-hydroxyphenylpropionic acid

p-hydroxyphenylacetic acid +- p-ethylphenol

p-cresol -+ p-hydroxybenzoic acid + phenol

These substances are all excreted in normal human urine (535) and are thought probably to arise from bacterial action, but not all the transformations postulated by Baumann are probable. For example, as we shall see below, phenol is formed directly from tyrosine. Though benzoic and p-hydroxybenzoic acids can be formed from phenylalanine and tyrosine, for example, by Pseudomonas species (346, 833), a plant rather than bacterial origin has been suggested for the benzoic acid moiety of the hippuric acid excreted by man (59).

Recent work suggests that benzoic acid can be formed in mammalian tissues (330a, 776a).

1 . Products Based on Decarboxylation and Amine Oxidation

Decarboxylation of amino acids is a typical feature of the bacterial decomposition of proteins. Both phenylethylamine and tyramine were isolated from putrid meat by Barger and Walpole (30), who considered it L‘extremely probable” that they were derived from phenylalanine and tyrosine, respectively. No cell-free preparation of phenylalanine decarboxylase appears to have been reported, but decarboxylation by a crude Streptococcus faecalis preparation provides a valuable method of phenylal- aiiine assay (887). Bacterial tyrosine decarboxylase has been studied in detail (495), especially by Gale and co-workers (summarized in 284). It requires pyridoxal phosphate as coenzyme (26, 326, 327) and, unlike mammalian tyrosine decarboxylase, also attacks dihydroxyphenylalanine. Decarboxylation normally only occurs in acid media and is considered primarily to be a protective mechanism tending to restore the pH to neu-

METABOLISM OF T H E AROMATIC AMINO ACIDS

ArH + NHa + CHa.CO.CO2H (e.g., phenol from tyrosine)

8-t yrosinaue T

dearninases or I

t ransaminases 1

Ar.CH2.CO.COsH - (e.g., phenylpyruvic acid)

A~.CI-IZ.CH,C>O?H 1

(o.g., I)hetiJ’lethyl:irniiic.

:tmirie oxidasr I

I Ar . CH 2. CBO

(e.g., phenylacetaldehyde) 1

> Ar.CH2.C02H (e.g., phenylacetic acid)

OH (e.g., phenylluctic acid)

?I > Ar.CHz.CH?.COzH ---+Ar. CH: CH. C02H ?

(e.g., cinnamic acid) (e.g., phenylpropi oriic acid) Diagram 16. Interrelationships of some side-chain metabolic reactions used by

microorganisms. Ar can be phenyl, p-hydroxyphenyl (as in tyrosine), indol-3-yl- (as in tryptophan), 3,4-dihydroxyphenyl, etc. Names given above are in general for substances derived from phenylalanine.

trality. Under alkaline conditions splitting of tyrosine to phenol, described later, is favored (495).

The amine resulting from decarboxylase action can be oxidatively de- amiriated to give the aldehyde or, more usually, the acid by further oxidation :

This reaction has been primarily studied in higher organisms (review 77) but probably occurs equally in microorganisms and also in plants, where it is probably intimately connected with biosynthesis of alkaloids ( q . ~ . ) .

It.CHzNH2 -+ Ilt,CH:NH] --t RCHO -, It.COrH

2. Products Based on Oxidative Deamination or Transamination Both reactions give the corresponding keto acid.

has been studied by, e.g., Stumpf and Green (839). Oxidative deamination

For cases of trans-

78 C . E. DliLGLIESH

amination see, e.g., references 739, 912. The keto acid can be further oxidized to the acetic acid derivative (the same as is formed by the decarboxylase-amine oxidase pathway).

Thus the following pathways have been demonstrated in a Vibrio species (167):

phenylalanirie -+ phenylpyruvic acid -+ phenylacetic acid

tyrosine ---f p-hydroxypheriylpyruvic acid --+ hornogentisic acid 1 L

1 furthcr metabolism

Reduction of the pyruvic acid would give the lactic acid (214, 50’21, and dehydration of this would give the acrylic acid (p-coumaric acid from tyrosine (e.g., 391) and cinnamic acid from phenylalanine). However, the acrylic acid derivative might be formed directly from the amino acid (cf. the direct conversion of histidine to urocanic acid). Reduction of the acrylic acids might be the origin of the propionic acid derivatives sometimes encountered (e.g., 100).

3. Phenol Formation from Tyrosine. p-l’yrosinase

Tyrosine is converted to phenol by an enzyme, p-tyrosinase, studied especially by Japanese workers (456, 654, 658, 879). The enzyme, which has been partially purified, is inhibited by carbonyl reagents and is dependent on pyridoxal phosphate. The reaction is mechanistically probably (593) very similar to the tryptophanase reaction and is discussed when considering the function of pyridoxal phosphate (p. 91).

4. Degradations Involving Opening of the Aromatic Ring

The microbiological degradation of many substances allied to the aromatic amino acids has been studied (reviews 346, 825). Thus the ring of catechol is opened to give ultimately p-ketoadipic acid (469) via cis-cis- muconic acid (246, 813, 826) :

I various

stapes H&, ,CO,H C H?

Analogous reactions might occur with, for example, dihydroxyphenylalanine or its derivatives, (e.g., 568, 829).

METABOLISM O F T H E 4ROMATIC AMINO ACIDS 70

VII. TRYPTOPHAN DEGRADATION BY THE KYNURENINE-NICOTINIC ACID PATHWAY

Among the most remarkable features of tryptophan metabolism is the widespread use of a common pathway by a wide range of organisms. Thus many microorganisms growing on tryptophan as sole carbon source metabolize it by a route very similar to that used in man, animals, insects, and probably also to a considerable extent in plants. In this discussion, therefore, evidence leading to the elucidation of the pathway will be drawn from many fields, and more detailed consideration of the individual steps will cover a range of species.

The structures of the substances discussed can be found in diagrams 17, 18, 20, and 21.

1. Establishment of the Relation between Tryptophan and Nicotinic Acid Nicotinic Acid

It was only many years after the discovery of tryptophan that a plausible degradative pathway could first be outlined, but during this early period a few tryptophan metabolites were identified. The long-known (559) kynurenic acid (structure, diagram 20; cf. 408) was shown in 1904 to be derived from tryptophan (220), but the considerable amount of work on kynureiiic acid formation (reviewed by Neubauer, 637) gave few useful results. Neubauer (637), however, made the plausible (and correct) suggestion that i t was derived from o-aminobenzoylpyruvic acid (structure, diagram 20).

It was not till 1925 that Japanese workers (587) reported a substance, kynurenine, subsequently shown (507) also to be derived from tryptophan. Unfortunately the position was confused by the incorrect structure originally assigned to kynurenine, and the correct structure (diagram 17) was only established in 1942 by Buteiiandt and co-workers (127, 129). Mean- while Musajo (616, 617) isolated (from the urine of rats on a fibrin-based diet fed added tryptophan) a substance which he called xanthurenic acid and which he showed to be 4,8-dihydroxyquinoli1ie-2-carboxylic acid (diagram 20). Lepkovsky and co-workers (550) later found the urine of pyridoxine-deficient rats to contain a green pigment which was (551) an iron complex of xaiithureriic acid.

A plausible hypothesis a t this stage (about 1943) was that a sequence occurred : tryptophan -+ an intermediate -+ kynurenine + kynurenic acid. I t was thought that the intermediate between tryptophan and kynurenine might be the so-called a-hydroxytryptophan (for structure see p. 83), which had been obtained (917) on hydrolysis of phalloidine, a toxic peptide from the fungus Amanita phalloides (567) ; such a pathway received

80 C. E. DALGLIESH

further support from the study of insect eye-pigments. From genetic experiments (reviews: I 16, 467) with mutants of Drosophila melanogaster and Ephestia kuhniella the brown eye-pigment, ommochrome, was known to be derived from tryptophan by the following sequence: tryptophan 3

V+-substance -+ rn+-substance ---f ommochrome. The V+-substance was identified as kynurenine (125, 857), and hydroxytryptophan was thought to act as a “prokynurenine” (126). Metabolic experiments with animals had meanwhile suggested that L-, rather than D-, tryptophan was precursor of the known metabolites and also suggested that an abnormal excretion of these metabolites occurred in pyridoxine deficiency (for further details see an earlier review by the author, 170).

I n 1945 Elvehjem and co-workers (518) reported that nicotinic acid- deficient rats would grow if given tryptophan, suggesting conversion of tryptophan to nicotinic acid. Rosen and co-workers (731) showed that administration of tryptophan to rats increased the urinary excretion of nicotinic acid derivatives, and numerous workers confirmed the conversion of tryptophan to nicotinic acid in man (399, 667, 755) and many other species (summary, 820). In the last ten years there has been intensive investigation of tryptophan metabolism.

2 . The General Outline of the Pathway

It was quickly established by many techniques (381, 430, 782, 817, 957, and review 170) that the conversion of tryptophan to nicotinic acid occurred in body tissues and was not due (except perhaps in part in excep- tional circ*mstances; cf. 170) t o intestinal bacteria. Moreover nutritional studies showed that kynurenine was probably also a precursor of nicotinic acid (457) and that kynurenine and xanthurenic acid excretion were increased in pyridoxine deficiency (21).

The probable course of tryptophan-nicotinic acid interconversion first became clear from experiments with microorganisms, mainly due to Mitch- ell, Nyc, Bonner, and their co-workers. In 1947 it was found (41) that a Neurospora mutant requiring tryptophan or nicotinic acid could equally

oxidsse Unknown tryptophan peroxidnse intermediate ___*

Formylkynurenine K ynurenine

Diagram 17. Reactions involved in transformation of tryptophan to kynurenine.

METABOLISM OF THE AROMATIC AMINO ACIDS 81

utilize L-kynurenine, and converted excess kynurenine to nicotinic acid. It was thought that nicotinic acid might be derived from the pyridine ring of kynurenic acid, but this was excluded (603) by tests with various potential oxidation products, and it was shown that the 8-hydroxy group in xanthurenic acid was in all probability introduced before cyclization. This immediately suggested 3-hydroxykynurenine (diagram 18) and the derived 3-hydroxyanthranilic acid as intermediates. Hydroxyanthranilic acid (651) was found to be rapidly converted to nicotinic acid, and was also accumulated by another Neurospora mutant (90, 92).

Hydroxykynurenine was isolated from the pupae of CaEliphora erythro- cephala (115, 623) and was identified as the cn+-substance referred to above, and its constitution was proved by synthesis (128, 500, 623). It was also obtained from the larvae of the silkworm Bombyx mori (392) and was shown to give nicotinic acid in a Neurospora mutant blocked after the kynurenine stage (970), which accumulated Na-acetylkynurenine (diagram 20) in the medium.

Both isotopic and nutritional experiments showed that the pathway established in microorganisms applied equally to mammals. Thus hydroxyanthranilic acid was converted to nicotinic acid (9, 604), which it could replace as a growth factor (944), whereas there was no similar conversion of anthranilic acid (343). An outstanding series of isotopic experiments, especially by Heidelberger and co-workers, showed that the @-carbon atom of the tryptophan side chain became the ,&carbon atom of the kynurenine side chain and that the side chain was lost in conversion of kynurenine to nicotinic acid (369, 371, 427). Moreover the carbon in the 3-position of the indole nucleus became the carboxyl carbon of nicotinic acid (370; this experiment proved conclusively the reality of the tryptophan-nicotinic acid conversion) and the indole nitrogen appeared with only slight dilution in kynurenine, kynurenic acid, and xanthurenic acid (759). All these relations are those to be expected for the pathway tryptophan -+ kynurenine -+ hydroxykynurenine (or its phosphate) 3 hydroxyanthranilic acid (or its phosphate) + nicotinic acid, illustrated in diagrams 17 and 18.

By 1950 the outline of the main pathway for tryptophan metabolism was therefore established, and it was becoming apparent that the over-all conversion of tryptophan to nicotinic acid was markedly reduced in many B-vitamin deficiencies. Thus tjhis occurred in pyridoxine deficiency (50, 387,732,784), riboflavin deficiency (387,455, G75), and thiamine deficieilcy (455, 675) but not in pantothcnate or folic acid deficiencies (455).

In the succeeding sectlions the various transformations involved will 1)e considered in detail.

Interest then moved to animals.

M E3

Kynurenic acid, xanthurenic acid, and derivatives (diagram 19)

CO.CHz.CH.COzH CO .CHz*CH*COzH I h I

NHz NHz

O'POaHs

PiHz

OH Kynurenine Hydrosxkynurenine Hydroxykynurenine phosphate ?

Anthranilic acid Hydrosyanthranilic acid Hydrosyanthranilic acid phosphate

(cf. intermediate Unknown diagram 21) * Xicotinic acid

Diagram 18. Transformation of kynurenine to h\droxynnthmnilic acid or i t s phosphate.

METABOLISM OF THE AROAlATIC AMINO ACIDS 83

3. The Converszon o j Trgptophan lo Formyllcynurenzne and Kynurenine

Details of the mechanism of tryptophan-kynurenine interconversion are largely due to Knox and his co-workers, in work originally arising from a wartime study of the quininc-oxidizing erizyine (477). In 19-19 it was reported (492) that kynurenine was formed from tryptophan by liver hom*ogenates under aerobic c*onditions, and detailed studies were published soon after (490, 591). L-Tryptophan was converted to kynurenirie by a highly specific enzyme contained in liver hom*ogenates of all species tested. The over-all reaction involved uptake of one molecule of oxygen, with libera- tion of one molecule of formic acid, but 110 carbon dioxide. The system was separable into two fractions. The first fraction converted tryptophan to a substance identified as formylkynureriine (diagram 17), and the second fraction contained another highly specific enzyme, formylase, which converted the formylkynureriine to kynurenine and formic acid. The enzyme system in the first fraction was itself divisible into two components. The first enzymic component required hydrogen peroxide generated in situ, added peroxide being iiieff ectivc, and converted tryptophan to an unknown intermediate, A. This was converted by the second enzymic component to formylkynureiiine with simultaneous formation of peroxide, which was then utilized by the first component. The three stages are therefore as folloms :

+ A 1 Tiyptophan + H D 2 ~

peroxidtrse

A oxidase

3 Formylkyriurenine - kynurenine + H COIH

+ o2 formyllipureii ine + I-1?01

formylase

and the first two of these stages are coupled t o form the tryptophan pcroxidase-oxidase system. Differences exist between tryptophan peroxidase and other peroxidases, especially in their relation to iron and copper (479, 481). ria (e.g., 366) and plants (e.g., 932).

The work already described suggested that it was a-hydroxytryptophan (more correctly described as oxindolylalanine, 157), which chemically is a plausible precursor of formylkynurenine (cf. review, 170).

Similar enzyme systems occur in ba

The nature of the intermediate A aroused widespread interest.

a-Hydroxytryptophan Oxindolylalanine

Rut when synthetic oxindolylalanine became available (156, 499) it8 was

84 C . E. D.\LGLIESH

OH OH

Possible dial intermediate Witkop’s proposed intermediate (0- hydroxy-$-tryptophan)

quickly shown iiot to be a tryptophan metabolite (11 , 117, 176, 584, 743). Alternatively hydrogen peroxide might add to tryptophan to give a diol (176, 490). This has not yet been synthesized and so cannot be tested i n biological systems (nor has the intermediate, A, been isolated),

From a consideration of numerous oxidations, using different types of reagent, of a variety of indoles and indole alkaloids Witkop arid co-workers concluded (216) that intermediate A was most likely to be of the reactive indolenine type (i.e., having a double bond between nitrogen and a-carbon of the pyrrole ring). They considered as the most likely substance P-hydroxy-$-tryptophan (structure above), which they proposed to synthesize, though no synthesis appears yet to have been reported. P-Hy- droxy-+-tryptophan might be converted to formylkynurenine either directly or by addition of water to give the previously proposed diol, which might then undergo dehydrogenation and ring-opening. A third possible route was considered, by way of the more highly oxidized hydroperoxide, but was thought to be less likely for an in vivo reartion. Indirect support for this third route, however, derives from work of Weiss and co-workers (442), who studied the breakdown of tryptophan 011 X-irradiation of aerated solutions. Such conditions bring about reactions analogous t o biological degradations in a large number of cases, and in agreement with this, formation of formylkynurenine from tryptophan was observed. The reaction required molecular oxygen, providing strong eviderire for a hydroperoxide type of intermediate which, for mechanistic reasons, they eon- sidered to have the hydrated structure depicted ahove rather than the indolenine structure considered by Witkop.

Possible hydroperoxide intermediate Weiss’s proposed interniediatc

The structure of the first intermediate in the normal biologiral degradation of tryptophan is therefore still uncertain. The difficulties in synthesizing any of the proposed intermediates are very considerable, and till such synthesis is accbomplished conclusive evidence will not be availablr. The problem is made somewhat less urgent by the fact that, whatever the

METABOLISM OF THE AROMATIC AMINO ACIDS 85

intermediate may be, no evidence (except possibly 310) has apparently yet been reported for its separate existence in vivo for any appreciable length of time.

No cofactors have been reported in the tryptophan peroxidase-oxidase reaction, but the marked reduction ill the conversion of tryptophan to nicotinic acid i n thiamine deficiency has been found in al! probability to be due to interference with the reaction at the tryptophan peroxidase- oxidase stage (173; cf. diagram 19). The evidence is still inadequate to show how enzyme function and vitamin are related. Biotin may also be concerned in the reaction (800; but see 175a).

The enzyme called formylase by Knox and Mehler (490, 591) arid kynurenine formamidase by Jakoby (437) is present in liver in a considerable excess relative to tryptophan peroxidase-oxidase (e.g., 491), and formylkynurenine is therefore not normally found in tissues or excreted in urine (e.g., 171). Partially purified tryptophan peroxidase-oxidase, from which formylase activity has been removed, accumulates formylkynurenine, shown (591) to be identical with synthetic (947 or better, 172) material. Formylase occurs widely in bacteria, and has been partially purified from Neurospora (437). In both higher and lower organisms the enzyme shows considerable specificity.

4, Tryptophan Peroxidase-Oxidase Adaptation

Although enzymic adaptation has been known for some time in microorganisms, tryptophan peroxidase-oxidase was probably the first animal enzyme to be proved to be adaptive. Knox and Mehler showed that if tryptophan is given to an animal orally, subcutaneously, or intraperi- toneally the liver tryptophan peroxidase-oxidase activity can rise up to ten-fold (491), and similar adaptation occurs in liver slices but not in hom*ogenates. These results have been confirmed by many workers (e.g., 210, 536, 537), and investigation of adaptive formation of the enzyme, which probably occurs by de novo synthesis of enzyme protein rather than by rearrangement of existing protein, has been used as an approach to the problem of protein biosynthesis (314a, 535a, 536, 537). Adapta- tion also, as might be expected, occurs in bacteria (366).

Tryptophan is, however, not the only agent which can bring about an increase in tryptophan peroxidase-oxidase. A smaller effect can be produced by substances which initiate the stress reaction of the adrenal- pituitary system (478). High X-irradiation produces a similar effect in normal, but not in adrenalectomized, animals (869). Cortisone reverses the effect of adrenalectomy (868), and glucocorticoids (e.g., cortisone and hydrocortisone) can themselves cause an increase in the enzyme (484). How these changes are brought about is still obscure; their elucidation

8 0 C. E. DALGLIESH

should give valuable information on the normal mechanism of adjustment of physiological processes.

6. Conversion o./ K!inureninc to tly~roxijkyrL~irciLine. IZole oJ IZibofEavivi

Experiments with mutants of microorganisms and insects, which made it likely that hydroxykynurenine is an intermediate in tryptophan nietab- olism, have been described above. Further evidence for the occurrence of hydroxykynmenine in insect larvae has since heen reported (575, 84-1, 845), and its relation to eye-pigments is discussed below. It is also formed in plants (932). Strong support for participation in manimaliari metabolism was provided by its identification in mamrrialiati urine (171, 176, 292, and c.f. further discussion later).

No enzyme system which will oxidize kyiiurenine to hydroxykynureriirie has yet been isolated in a cell-free state from any species, and there is some evidence that direct conversion may not normally occur, a t least in mammals. Riboflavin tvas suggested to be concerned in hydroxykynurenine formation a t a comparatively early stage (387), and this has been supported by nutritional experiments.

Tryptophan metabolism can be briefly represented as in diagram 19.

Tryptophan' Iiynurenic acid Xanthurenic acid and conjugated and conjugated derivatives of derivatives of

c __- - - - - - - - c kynurenine hydroxykynurenine

t B I

A 4 t I A _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ A

Hydroxyanthranilic acid Anthranilic acid

1 Formylkynurenine - Kynurenine _f__c Hydroxykynurenine

Diagram 19. Relation of vitamins to tryptophan metabolism. Deficiencies of pyridoxine, riboflavin, and thiamine cause blocks a t A - - - A , I3 - - - H, and (' - - - P , respectively.

It has been shown by the author that examination of the products excreted after administration of tryptophan to vitarnin-deficient animals can give valuable information on the function of that vitamin in tryptophan metabolism (142, 171, 173). When tryptophan is given to the riboflavin- deficient rat there is a large excretion of those substances which lie to the left of line BB in diagram 19 (142, 582). This clearly indicates that this is the step a t which riboflavin functions, and this is strongly supported by the fact that riboflavin deficiency can reduce up to ten-fold the conversion of t,ryptophan t,o quinolinic acid, whereas similar conversion of hydroxy- kynureiijrie is unaffected (385). On the other hand, the excretory pattern

iMRT.\BOLISM OF THE AltOMSTIC .\MINO hCIDS 87

obtained after feeding tryptophan to riboflavin-deficient rats is not clean cut as therc is also an increase in the excretion of xanthurenic acid, which lies to the right of line BB in diagram 19 (142). A possible reason for this emerged from work of Wiss and Hellniann (375, 942), who found that tryptophan and kynureninc were converted by liver hom*ogenates not to 3-hydroxyanthranilic acid but to a derivative, identified as its O-phosphate, which also arts as a nicotinic acid precursor. Both kynurenine and hydroxykyriurenine are split by an enzyme, kynureninase (considered in more detail below), to give anthranilic acid and hydroxyanthranilic acid, respectively. As hydroxyanthranilic acid was not converted by liver to the phosphate, it is reasonable to suppose that the hydroxyanthranilic acid phosphate was formed by action of kynureiiinase on hydroxykynurenine phosphate (structure: diagram 18). This would indicate that the hydroxylation stage in tryptophan metabolism is an oxidative phosphoryla- tion rather than a simple oxidation, energy from this phosphate bond possibly being concerned in subsequent conversion of hydroxyanthranilic acid to nicotinic acid.

Urinary excretion of hydroxykynurenine, rather than its phosphate, is not surprising in view of the widespread occurrence of phosphatases in many organs including the kidney. Riboflavin might therefore be con- verned with a phosphorylative rather than a purely oxidative function, and the increased xaiithureriic acid excretion in riboflavin deficiency can be plausibly explained on this basis (142). It is of interest that in Knox's early work on the yuinine-oxidizing system of liver (477) the partially purified enzyme which hydroxylated quinolines appeared to be a flavoprotein.

In view of the current interest in hydroxylation reactions in vivo i t is to be hoped that this stage of tryptophan metabolism mill receive more detailed attention from enzymologists. At present it can be stated definitely only that kynurenine (or possibly a derivative such as N"-acetylkynureniiie; 170, 173) is converted either to hydroxykynureiiine or to some simple derivative a t the same level of oxidation. It is of interest that both kynurenine and hydroxykynurenine are formed on photooxidation of tryptophan, especially in the presence of ferrous iron (972).

6. Hgdroxgkynurenine and Insect Eye Pigments

The identification of hydroxykynurenine as the cn+-substance which is the precursor of insect eye pigments (ommochromes) has already been described. Ommochromes had previously been considered to be pterin derivatives (140, 534), but there is no obvious route for the conversion of hydroxykyriurenine to the pterin type of structure. However, pterins also occur in insect eyes (275). More recent work has shown that the eye pig-

88 C. E. DALGLIESII

ments, which appear to be similar to the pigments of insect eggs, are fornicd directly from hydroxykynureiiine to give derivatives forming complrxcs with metals, especially copper arid iroii (468). In the eggs the pigmc3iit seems to serve a definite physiological function, as pigmented eggs arc more viable than noiipigmented (467). Hydroxykynurenine also seems to participate in the hardening of the insect cuticle (678).

The chemical nature of the pigments is becoming evident from work in Butenandt's laboratory, which has eliminated the pterin type of structure. Ommochromes can be divided into ommins, of high molecular weight, and ommatins, of low molecular weight. Various ommatins were isolated (122) and studied chemically (120) and shown by comparison with model compounds (121) probably to be phenoxazine derivatives. Thus two molecules of hydroxykynurenine can react together to give a phenoxazine as follows (where R is the side chain .C0.CHz.CH(NH2) vCOnH):

R R R R

__c f 2 H

013 OH Hydroxykynurenine phenoxazine derivative

transamination and cyclization of one side chain J

The phenoxazine can then participate in reversible oxidation-reduction reactions, and moreover by transamination and cyclization of one side chain, in a manner analogous to xanthurenic acid formation discussed below, can give a pyridinophenoxazine derivative which equally undergoes reversible oxidation-reduction, and which, being a derivative of 8-hydroxyquinoline, would bind metals strongly (see also 122a).

7. Kynureninase, Kynurenine l'ransaminase, and the Formation of Anthranilic, Kynurenic, Hydroxyanthranilic,

and Xanthurenic Acids

Reference has already been made to the abnormally high excretion of kynurenine and xanthurenic acid in pyridoxine deficiency. This was confirmed by many workers (e.g., 372, 599, 620, 674, 698, 732), and the reason for it became evident from enzymic experiments.

The first mention of enzymic conversion of kynurenine to kynurenic

METABOLISM OF THE AROMATIC AMINO ACIDS 89

acid and anthranilic acid was by Kotake (501, 512), the enzyme forming anthranilic acid being named kynureninase. Braunshtein and colleagues (102) showed that alanine was also formed, and that the enzyme occurred in the livers arid kidneys of all species tested. It was inhibited by carbonyl reagents and was dependent on pyridoxal phosphate. The amount of enzyme was markedly reduced in the liver of pyridoxine-deficient rats, but the pyridoxine deficiency affected only the degradation of kynurenine and not its formation from tryptophan. Kynureninase activity was restored to normal on adding pyridoxal phosphate. The formation of anthranilic acid and alanine was confirmed by Wiss (936, 941, 945) and the pyridoxal phosphate requirement by Knox (176). The enzyme attacks kynurenine and hydroxykynurenine at comparable rates (176, 480, 940) and is unspecific in the sense that it will attack (at varying rates) many substrates containing the .CO.CH2.CH(NH2).COzH side chain (123, 124, 376, 939, 940). Powerful support for these enzymic results was obtained from nutritional experiments (171, 176, 293) which showed that in pyridoxine deficiency tryptophan gave rise to urinary excretion of many metabolites besides the previously known kynurenine, kynureriic acid, and xanthurenic acid. The majority of these were identified (171), and all still carried the carbon skeleton of the original tryptophan side chain. It was therefore apparent that pyridoxine functioned in tryptophan metabolism at the stage a t which the original side chain was removed. Knox et al. (176) also found that after prolonged pyridoxine deficiency kynureninase activity of the liver is not restored by adding pyridoxal phosphate in vitro, and this, too, is in accord .with results of nutritional experiments (732, 768). As pyridoxine deficiency has no effect on conversion of tryptophan to kynurenine (102) or of hydroxyanthranilic acid to nicotinic acid (387), it must participate in tryptophan metabolism as indicated in diagrams 18 and 19.

Knox et al. (176) found that formation of anthranilic acid from kynurenine was always accompanied by formation of kynurenic acid, and they considered that the keto acid (0-aminobenzoylpyruvic acid, diagram 20) might be a common intermediate in formation of both substances. This was soon disproved, and it is now clear that two independent reactions are involved, as illustrated in diagram 20.

Wiss (937, 938) fractionated crude liver extracts to give a kynureninase fraction which would form anthranilic acid, but not kynurenic acid, and a transaminase fraction which mould not form anthranilic acid, but formed kynurenic acid provided an a-keto acid was present. o-Aminobenzoyl- pyruvic acid, the keto acid corresponding to kynurenine, is known to cyclize spontaneously to kynurenic acid (622), and the absence of ammonia production and requirement for an a-keto acid (cf. also 434) suggests that

00 C . E. DALGLIESH

OH [p CO.CH2. CO * COZH 1 spontsneoua

_____)

NHz N' COzH R It

L J

o- Aminobenzoylpyruvic acid Kynurenic acid (R- H) derivatives Xanthurenic acid (R- OH)

kynurenine transaminase I

f CHJ*CH*CO~H I

' NHz NHz

kynureninase

(cofactor:

CO *CHI* CH *COzH I 8: KH2 NH2 pyridoxal phosphate)

R R Kynurenine (R * H)

Hydroxykynurenine (R - OH) , An thranilic acid (R H) + alanine Hydroxyanthranilic acid (R = OH)

Conjugated derivatives of h ydroxy kynurenine

I NH * CO * CHI

q C 0 *CHz*CH. COzH

NHz R

X@-Acetylkynurenine (RE H) Glucuronide (R - 0.C6HPOD) Sulfate (R= O*SO,H) Na-Acetylhydrosykyn(Irenine (R = OH)

Diagram 20. Interrelationship of derivatives of kynurenine and hydroxykyna- renine.

it is formed by transamination alone and not by oxidative deaminatioii. 3-Hydroxykynureniiie undergoes similar splitting (kynureninase) and cay- clization (transaminase) reactions. Pyridoxal phosphate is coenzyme for both reactions, mid comparison of activities of various fractions made it clear that the same enzymes attack both kynurenine aiid hydroxykynurc- nine (938). These results have been confirmed by Knox (480), who showed that under normal circunistances the formation and degradation of kynurenine in the liver proceed at comparable rates. Iiynurenine and hydroxykyiiurenine are also metabolized by both kynureninase and kynurenine transaminase in the kidney. But whereas kynureninase activity of kidney is only about one-tenth that of liver (480), the transaminase activity of kidney is much higher (583), aiid kynurenine is therefore converted almost exclusively to kynurenic acid by kidney preparations (cf. 583a, 585).

The detailed mechanism of pyridoxal phosphate participation in the kynureninase and kynurenine transaminase reactions is considered in detail later. Of interest in this connect,ion is the finding that other amino

METABOLISM O F THE AIEOMATIC AMINO ACIDS 91

acids inhibit mammalian kynureninase (306). This is probably due to the requirement for pyridoxal phosphate oE most reactions involving amino acids, and resultaut competition for the coenzyme. Such competition probably explains, a t least in part, the interference with tryptophail metabolism observed 011 giving excess of threonine, or in general amino acid imbal- ance (12, 315, 384, S l l ) , which has been attributed to interference with tryptophan absorption from the gut and to preferential use of tryptophan for protein biosynthesis.

Microorganisms also utilize both the kynureninase and kynuren ine transaminase reactions. Bacterial kynureninase has been partially purified and, like the mammalian enzyme, requires pyridoxal phosphate (367) and givs anthranilic acid and alanine, but not pyruvic acid (600). Moreover bacterial kynureninase has no transaminating activity (GOO), and bacterial kynurenine transaminase has no kynureninase activity (601). The kynureninase of Neurospora has been studied in some detail (438, 433). This again resembles the mammalian enzyme in attacking both L-kynurenine and 3-hydroxy-~-kynurenine (it also attacks formyl-L-kynureniiw, but not D-kynurenine or N"-acetyl-L-kynurenine) and in requiring pyridclxal phosphate. Magnesium also activates the system. The Neurospora enzyme is also inhibited by amino acids and amines, probably by competitive removal of pyridoxal phosphate.

Xanthureriic acid excretion can be caused in the rabbit by a vitamin-E as n.ell as vitamin-Be-deficiency (200). This is more likely to be due to vitamin interaction than to a direct effect of vitamin E on tryptaphan metabolism.

8. Mechanism of Action of Pyridoxal Phosphate in Reactions I7i volving Aromatic Amino Acids

Pyridoxal phosphate is the coenzyme in a large number of amino acid reactions. At this point i t is convenient to consider together the mechanism of those pyridoxal-dependent reactions concerned with aromatic amino acids. The reactions concerned are (1) keto acid formation (e.g., from kynurenine, above), (2) decarboxylation (e.g., of 5-hydraxytrypto- phan to 5-hydroxytryptamine, p. loo), (3) scission of the side chain (eg., p-tyrosinase, p. 78; tryptophanase, p. 110; and kynureninase, above), and (4) synthesis (e.g., of tryptophan from indole and serine, p. 40). Many workers have considered the mechanism of one or more of these reactions (e.g., 24, 216, 361, 595), but a unified theory is primarily due to Snell and his colleagues (summarized in 593). Snell's experiments have been carried out largely in vitro, and it should be emphasized that in vivo it is the enzyme protein which probably directs the clectromeric changes.

92 C. E. DALGLIESH

The amino acid, coordinating metal, aud pyridoxal phosphate are considered to react to give a complex having a conjugated system of double bonds extending from the electron-attracting nitrogen of the pyridoxal to the site of reaction. The coordinating metal probably functions both by promoting Schiff’s hsse formation and by maintaining planarity in the conjugated system through chelate-ring formation. In the following diagram [PI represeiibs the phosphate group of pyridoxal phosphate which com- bines with the apoenzyme (cf. 300) :

Aromatic amino acid Coordinating metal Pyridoxal phosphate or its complex with

the apoenzyme

Resonance forms of the coenzynie ainiiio acid complex, A; electromeric displacements indicated by the curly arrows.

The complex may then split in various ways. If splitting occurs a t aa, keto acid formation can occur by way of the following prototropic changes:

The keto acid and pyridoxamine phosphate result, and the latter can

METABOLISM O F THE AROMATIC AMINO ACIDS 9 3

then transaminate by a reverse series of changes with another a-kc:to acid. The pyridoxal phosphate thus acts essentially as a nitrogen carrier between amino acid and keto acid, and the reactions may well be linked so that pyridoxamine phosphate as such has no independent existence.

If, however, the complex breaks a t point bb in the first diagram above, decarboxylation results as follows :

Complex A, above amine + COz+ pyridoxal phosphs te 4

The above reactions are common to most amino acids. In the case of aromatic amino acids the aryl group (except for the unactivltted phenyl group of phenylalanine) can itself act as an electron-attracting group. If this occurs the aryl group can be split off by the following series of rertctions:

Scission of the side chain leaves an Ar- ion which takes up a proton to give indole from tryptophan (as with tryptophanase) or phenol from tyrosine (as with @-tyrosinase). The side chain of the original molecule is left as the pyridoxal phosphate complex of aminoacrylic acid, and on hydrolysis the aminoacrylic acid tautomerizes to the imine of pyruvic acid which is hydrolyzed to pyruvic acid and ammonia:

CHz=C*COzH --+ CHs.C.COzH + CHS.CO*COzH + NH3 I

NHz I I NH

Although the kyriureriinase reaction involves a scission, i t is probably of

94 C . E. DALGLIESH

a differmt type. The Schiff’s base from kynurenine arid pyridoxal phosphate is an imine of a p-diketone, aiid i t is to be supposed that the enzynw probably carries out hydrolysis analogous to the ready chemical hydrolysis of such cwmpoundtj. But the intermediacy of the Schiff’s base is not universally accepted (439), and it is significant that the side chain iii this rcw- tion is liberated as alauine, aiid not as pyruvic acid and ammonia.

It has also been suggested that the result can be explained by a scission of the tryptophanase type, together with an internal oxidation-reduction to give the observed products (561.a).

The pyridoxal phosphate complex of aniiiioacrylic acid can also be formed from seriiie by loss of an OH- radical in a manner aiialogous to loss of the Alir radical depicted above. This complex contains a reactive double bond to which the reactive @-hydrogen of indole can add, giving a complex which on hydrolysis yields tryptophan. Such a mechanism is in accord with the known facts on tryptophan hiosynthesis (cf. 858, and previous discussion, p. 41).

9. Excretion of I lydroxykynurenine and Xanthurenic Acid by M a n

Excretion of hydroxykynurenine or xanthureiiic acid by man has been coniiected with pathological states. Excretion of xanthurenic acid by py- ridoxiiie-defic.ient animals was observed a t an early stage, as already described. Xanthureiiic acid is also excreted, especially after a loading dose of tryptophan, during pyridoxine deficiency in man, whether this is produced by a true dietary deficiency (311) or by administration of the vitamin antagonist deoxypyridoxine (294). Pyridoxine deficiency in man caii to some extent be judgod by determining (599, 621, 732,733, 898) the amount of xanthurenic acid exrreted after a standard dose of tryptophan, the value so obtained being known as the xanthurenic index (145). il high xaiithu- renic index is found in pregnancy (155a, 618, 891, 899), when the extra demand for the vitamin may cause a “physiogical” pyridoxine deficiency. This excretion caii be reduced by pyridoxine administration (e.g., 900). An especially high xanthurenic index may be found in certain disorders of pregnancy (823, 899). After giving a loading dose of tryptophan in pregnancy not only xanthurenic acid is excreted, but also (901) the whole range of tryptophaii metabolites observed in the pyridoxine deficient rat (cf. 171).

Further interest was aroused when Kotake (503) claimed that xanthurenic acid would cause diabetes, that it occurred in diabetic urines (515), and that diabetes induced by xanthurenic acid was reduced by insulin administration (509). Further supporting work has since been published from the same laboratory (50-1-506, 508), but German workers (400) have been quite unahle to demonstrate any diabetogenic action of xanthurenic acid,

METABOLISM OF THE AROMATIC AMINO ACIDS 95

except for a transient hyperglycemia occurring when xanthurenic acid administration was superposed on a pyridoxine deficiency (908).

Hydroxykynurenine excretion in pathological states was also first reported by Japanese workers (573)) who identified i t as the substance causing the diazo reaction and the Weiss urochromogen reaction in urines from cases of severe tuberculosis. This was confirmed in the author’s laboratory (178), where i t was also shown that the excretion is unrelated to tuberculosis as such. Hydroxykynurenine excretion occurs in a large proportion of patients with fevers of varying etiology and is in all probability due to the increased rate of breakdown of body proteins in fever. Presumably the protein breakdown induces an adaptive increase in tryptophan peroxidase-oxidase, and the capacity of the available kynureninase, which comes laterinthe metabolic chain and is not an adaptive enzyme (480), is exceeded.

Hydroxykynurenine excretion in fever is sometimes accompanied by smaller amounts of kynurenine, whereas if excess tryptophan is taken by mouth large amounts of kynurenine, but negligible hydroxykynurenine, are excreted. These differences in metabolism of exogenous and eiidogenous tryptophan are unlikely to be due to use of different metabolic pathways and suggest that tryptophan molecules from endogenous protein breakdown and from exogenous sources do not necessarily equilibrate in a common body tryptophan “pool.”

Hydroxykynurenine and kynurenine excretion in leukemia (618, 619) and in diabetes (515) have also been reported. In both cases fever is the probable cause. Diabetes as such does not result in hydroxykynurenine excretion (178), which probably occurs only when there is fever due to complications. Traces of kynurenine may also be encountered in normal urine (8 18a).

For recent work on kynurenine and hydroxykynurenine excretion see (6194.

10. S ide lieactions o j Kynureninc, Hydrozyliynurenine, Anthranilic A c i d , and Hydroxyanthranilic Acid

Na-Acetylkynurenine (i.e., acetylated on the aliphatic amino group) was isolated from a Neurospora mutant culture (970) and was also found in the urine of pyridoxine-deficient rats together with the analogous Nu-acetylhy- droxykynurenine (171). It is possible, though there is no direct evidence, that these N‘-acetyl derivativcs play some part in normal tryptophan mc- tabolism in the rat (ef. 170, 173)) or they may, as is probably the case in Neurospora, merely be products of side reactions occurring in the presence of abnormal amounts of kynurcnine and hydroxykynurenine. Hydroxy- kynurcniric is also cwreted by the pyridoxine-deficicnt rat as its O-sulfate

96 C. E. DALGLIESH

and 0-glucuronide (171). These are probably simple “detoxication” products without other metabolic significance. The formation of all these compounds is illustrated in diagram 20 above.

Xanthurenic acid if present in unusual amount is also treated as a foreign phenol and excreted in conjugated form (171, 513), though the nature of the conjugating groups has not been established. Xanthurenic acid can probably also be degraded to some extent by an enzyme, xanthurenicase, in liver and kidney (510), but the products have not been identified.

Kynurenic acid and xanthureriic acid are both metabolically speaking waste products in that they cannot be converted to nicotinic acid. The same applies t o anthranilic acid, which apparently cannot be directly converted to hydroxyanthranilic acid to any appreciable extent in mammals or even in microorganisms (9 1, 663). Under normal circ*mstances mammalian metabolism of tryptophan is so well regulated that kynureiiine is apparently converted to hydroxykynurenine (or its phosphate) as fast as i t is formed. As a result, under normal circ*mstances only hydroxykynurenine (or its phosphate) is a kynureninase substrate and therefore no anthranilic acid is formed. Supporting evidence is that in man and other mammals anthranilic acid taken by mouth is excreted either unchanged, as the glucuronide, or as the glycine conjugate, o-aminohippuric acid (103, 142, 582), though a small amount of 3-hydroxyanthranilic acid and 5-hydroxyanthranilic acid (514) may be formed, possibly by unspecific hydroxylation (cf. 174). If anthranilic acid were formed in normal metabolism at least part would therefore almost certainly be excreted. However, anthranilic acid excretion does not occur in man or mammals except after intake of excess tryptophan. It is of considerable interest that a rare human disorder, congenital hypoplastic anemia, is accompanied by anthranilic acid excretion (lo), indicating an associated anomaly in tryptophan metabolism.

Both anthranilic acid and hydroxyanthranilic acid call he formed from t ryptophan in insect mutants, and both arc conjugated with glyvine to give substituted hippuric acids (706), whereas in plants or bacteria anthranilic acid tends to be conjugated as the 6-glucoside (848).

Another interesting variant of tryptophan metabolism has been fouiid in silkworm pupae ( I 18), which form kyriurine (4-hydroxyquinoline). This might arise by direct decarboxylation of kynurenic acid, but it is more likely (1 19) that it is formed by decarboxylation of kynureiiine to kynurcn- aminc, which would be expected to give kynurine through the action of aminc oxidasc.

T-Iydroxykynureiiiiie can also be converted by mouse liver, probably via the corresponding amine, to 4: 8-dihydroxyquinoline (571a).

METABOLISM OF T H E AROMATIC AMINO ACIDS 9 7

11. Conversion of Hydroxyanthranilic Acid to Nicotinic Acid

The discovery that 3-hydroxyanthranilic acid is a precursor of nicotinic acid in Neurospora (YO, 516, 603) and the rat (9, 516, 604, 943, 944) has already been mentioned. It had been known for some time (810) that rats fed tryptophan excreted in their urine a substance which, after hcing autoclaved in acid solution, had nicotinic acid activity. This was identified by Henderson (379, 383) as quinolinic acid (structure, diagram 21). Mutants of Neurospora were also found which accumulated quinolinic acid, and this could be used by some, but not all, nicotinic-requiring Neurospora strains (94). In the rat quinolinic acid was mostly excreted unchanged, but part was converted to nicotinic acid, though much less efficiently than was hydroxyanthranilic acid (380). Hydroxyarithranilic acid was converted to quinolinic acid by liver slices (386)) and i t was considered possible that quinolinic acid was a normal intermediate in nicotinic acid formation. The lesser effectiveness as a nicotinic precursor of quinolinic acid relative to hydroxyanthranilic acid was attributed to the conversion of quinolinic to nicotinic acid being slou and rate-determining. At first the position was, moreover, complicated by isotopic experiments with NI6-ammonia and unlabeled hydroxyanthranilic acid in Neurospora (541). Iso- tope was incorporated in the nicotinic acid to a degree suggesting formation of a symmetrical intermediate. Sucl an intermediate was difficult to visualize, but the difficulty was later removed when the isotope incorporation was shown (971) to be due to simultaneous active growth of the organism. If no growth occurs the nitrogen of nicotinic acid is derived entirely from the nitrogen of hydroxyanthranilic acid. It is of considerable interest that recent evidence shows (968) that in 8. coli and B. subtilis, but not in Neurospora, nicotinic acid can be formed by a route not involving tryptophan.

The results with Neurospora led Bonner and Yanofsky (94) to suggest that the conversion of hydroxyanthranilic acid t o nicotinic acid went by way of Intermediates A and B of diagram 21. Quinolinic acid formation was thought to be a shunt or side reaction of intermediate A, slow conversion to nicotinic acid possibly providing an alternative pathway. A similar conclusion was drawn from experiments in the rat (971), and it is now generally agreed that the conversion of quinolinic acid to nicotinic acid is a t best of the order of a side reaction (e.g., 685,754, and in man, 397,696).

The conversion of hydroxyanthranilic acid to nicotinic acid in the rat has been shown unambiguously in isotopic experiments (344). Conversion to either quinolinic or nicotinic acids must involve open-chain intermediates such as A and B of diagram 21. Japanese workers (572) suggested that these were formed by scission between the catechol hydroxyls of the ring

08 C. E. DALGLIESH

3-Hydroxykynurenine +b 3,4-Dihydroxykynurenine

I (or its phosphate)

1 c-g: - HO QZ OH OH

3-Hydroanthranilic acid 3,4Dihydroxyanthranilic acid (or its phosphate)

COiH

Isocinchomeronic acid

1 2 stages \ /

Quinolinic acid . . . . . .

I I

I I ?

Intermediate A

OHC nCoiH NH,

Intermediate B

Nicotinic acid Diagram 21. The probable pathway (via Intermediates A and B), and other sug-

gested pathways, for conversion of hydroxyanthranilic acid t o nicotinic acid.

of 3,4-dihydroxyanthranilic acid (diagram 21), and i t was claimed that synthetic dihydroxyanthranilic acid gave rise to nicotinic acid (433, 572). However, many workers in enzymic and growth experiments on both animals and microorganisms could find no evidence that dihydroxyanthranilic acid was a nicotinic precursor (89,'376,382). Moreover, its presumed pro- genitor, 3,4-dihydroxykynurenine, has also been shown not to be B tryptophan metabolite (123, 171, 795).

Isotopic experiments (763) with tryptophaii labeled with NI5 and deuterium in the indole ring have shown that quinolinic acid nitrogen is probably entirely derived from the indole nitrogen of tryptophan, and that scissioii of the benzene ring probably occurs between carbons 3 and 4. Presumably, therefore, the hydroxyanthranilic acid is converted to intermediate A without participation of a catechol-type intermediate, and it is possible that the phosphate-bond energy of hydroxyanthranilic acid phosphate (if this is in fact an intermediate) may contribute to the transformation. It is known

XETABOLISM OF THE AROMATIC AMINO ACIDS 99

that phenols can be converted to ortho-quinonoid compounds without the necessity of passing through a catechol stage (946), and it may well be that such a quinonoid compound participates in the conversion of hydroxyanthranilic acid to intermediate A. This is supported by spectroscopic evidence suggesting that two intermediates, the first possibly quinonoid, are involved in the conversion of hydroxyanthrariilic acid to quinolinic acid (89). A quirioneimiiie structure has been suggested (608) for the first intermediate, but no attention appears to have been paid to the necessity for a fumaric ---$ rnaleic isomerizatiori of intermediate A (but cf. 938a).

An alternative route to nicotinic acid involves scission of hydroxyanthra- iiilic acid between carbons 2 and 3, with intermediate formation of isocinchomeronic acid (diagram 21). But the latter does not act as a nicotinic precursor in Neurospora (374), and this route can probably be excluded.

The enzyme (386, 971) converting hydroxyarithranilic acid to quirioliiiic acid has been widely studied. Rat liver hom*ogenates or acetone powder can carry out the conversion in yields of 73 % to 100 % (88), though an enzyme located in the heavy particles of the cell can also convert hydroxyanthranilic acid to a red-colored product (897). The quinolinic-forming system occurs widely in liver and kidney (676) but not in other organs (812), and the activity is associated with the nonparticulate fraction of an hom*o- geriate (812). The spectroscopic changes occurring in the reaction are referred to above. The enzyme shows a requirement for ferrous iron under certain conditions (564, G O S ) , and to some extent resembles the enzymes oxidizing hom*ogentisic acid, catechol, and protocatechuic acid (162).

It is remarkable that the intermediates in the conversion of hydroxyanthranilic acid to nicotinic acid are still not known with certainty. Inter- mediates A and B of diagram 21 are plausible suggestions, but no synthesis of either has been reported. Both are extremely unlikely to be stable in the free state, but should be obtainable as simple derivatives. In the free state Intermediate A, for example, might be expected to tautomerixe to the iniino acid, and hence give keto acid and ammonia, or it could cyclixe to a piperazine or t,o quinolinic acid. Tautonierism through the iniino acid would eliminate the necessity for a fumaric ---f maleic isomerization. It is quite possible that spontaneous cyclization explains the prominent part quinolinic acid plays in work 011 hydroxyanthranilic-nicotinic conversion. If the latter occurred in the following .way:

Hydroxyanthra- enzyme spontaneous

Intermediate + Quinolinic acid A I

(diagram 21) ! -4

nilic acid

-COa; enzyme B I I -coz ? .1

Intermediate spontaneous + Nicotinic acid B

(diagram 21)

100 C. E. DALGLIESH

and if eiizyme B vere more labile than enzyme A, or if the two enzymes became spatially separated on disorganization of the cell as in an hom*ogenate, then hydroxyaiithranilic acid mould be expected to give quinolinic acid. There is good evidence that the conversion of intermediate A to quinolinic acid is spontaneous (564, 590a). The low activity of quinolinic acid as a nicotinic precursor might be due to some much slower and less specific8 decarboxylation process.

If a large dose of hydroxyanthranilic acid is given to nu aiiinial n small proportion is excreted unchanged (104). Hydroxyanthranilic acid is also excreted by man after a large dose of tryptophan ((i96), and has been found in human urine in tuberculosis (624). The latter is probably due to high eridogeiious protein breakdown (cf. 178).

It is possible that quiiioliuic acid might he decarboxylated to picolinic (pyridine-karboxylic) acid as well as to nicotinic (pyridine-3-carboxylic) acid. Such may be the origin of the hoinarine (picolinic acid betaine) widely occurring in marine organisms (e.g., 423).

12. Tryptophan, Nicotinic Ac id , and the Pyridine Nucleotides

The pyridine nucleotides are the functional form of nicotinic acid, but still comparatively little work has been done on their relation to tryptophan- nicotinic acid metabolism. Two possibilities must be considered; the pyridine riucleotides may be formed from tryptophari without intermediacy of nicotinic acid and only give nicotinic acid on breakdown, or nicotinic acid first formed from tryptophan may be incorporated into pyridine nucleotides. The latter now seems the more likely possibility, though the former has not been excluded.

pyridine nucleotides ? further breakdown

nicotinic acid ------+ nicotinic metubolitcs

Injected tryptophan causes a rise in erythrocyte DPN in the rat (562). The problem was taken up by Elvehjem and his school (cf. review, 224), who at first found tryptophan to be more active than nicotinamide in stimulating synthesis of rat-liver DPN and TPN (924, 925). Nicotinamide had, however, a sparing effect in young, but not in adult, rats (925). In pyridoxine deficiency conversion of tryptophan to pyridine nucleotides

METABOLISM O F THE AROMATIC AMINO ACIDS 101

appeared to be unaffected (926) unless deoxypyridoxine were also given (519). These results suggested that nicotinic acid and amide were not, iiccessary intermediates, hut later work (254, 255) showed that tryptophan aiid nicotinic acid were equally good pyridine nucleotide precursors on a molar h i s (previous results having been due to use of a relatively much larger amourit, of tryptophan), whereas nicotinamide was more effi- cient than either. But i t was still felt that nicotinic acid was not a necessary intermediate. Conversion of nicotinic acid to pyridine nucleotides was, as is to he expected, unaffected by pyridoxine deficiency (519).

Other workers (206,533) have studied the pyridine nucleotides of human red blood cells, which are known to be increased after nicotinic acid administration (342, 394). Sarett (206) found that both tryptophan and nicotinic acid given orally to man cause a rise in blood pyridine nucleotides, the peak of the increase occurring after 10 hours with nicotinic acid and after 18 to 20 hours with tryptophan. Pyridine nucleotides can be synthesized by washed human red cells when incubated in Ringer phosphate with nicotinamide but not with tryptophan (159), and this is probably brought about by the enzyme, diphosphopyridinenucleotidase, studied by Colowick and his colleagues, which catalyzes exhange of the nicotinamide moiety of DPN with free nicotinamide (973) and also with related substances such as iso- nicotinic acid hydrazide (974). Improved methods of determining pyridine nucleotides and their precursors in blood (e.g. 520) should lead to further advances in this field (cf. 113b).

Bacterial synthesis of pyridine nucleotides may proceed somewhat differ- ently. Hughes (421) has produced evidence for the following pathway of cozymase synthesis:

1. nicotinic acid + nicotinamide 2, nicotinamide -+ nicotinamide riboside 3. nicotinamide riboside -+ nicotinamide ribonucleotide 4. nicotinamide ribonucleotide -+ cozymase

However, isotopically labelednicotinic acidreadily gives rise to labeled DPK and TPN attached to the outside of certain bacterial cells (95), suggesting that exchange reactions may also occur.

The biosynthesis, function, and degradation of the pyridine nucleotides have been reviewed by Singer and Kearney (812a).

13. Further Metabolism of Nicotinic Acid

In the greater part of the preceding discussion nicotinic acid has, for brev- ity, been treated as the end product of tryptophan metabolism. In fact the end products consist of several metabolites of nicotinic acid, which can

102 C . E. DALGLIESH

f lCO * NH * CHs* COnH _____c

QcoaH ” Nicotinic acid Nicotinuric acid

(nicotinyl glycine) I t c pyridine ------- nucleotides

Nicotinarnide

flco”H* ____c

1 QCO’”l”

I O 1 ; J Me Me

N-iJ4ethylnicotinamide 6-Pyridone

Diagram 22. Further metabolism of nicotinic acid in animals.

only briefly be considered here. The particular metabolites formed vary with the species. Failure to realize this fact has in the past led to many erroneous conclusions owing to failure to estimate a metabolite appropriate to the species investigated. On the whole herbivorous species tend to excrete nicotinic acid free or as a conjugate, whereas carnivorous and omniv- orous species tend to carry out N-methylation (668). But a herbivorous animal such as the guinea pig, which normally excretes nicotinic acid un- methylated, is said to carry out methylation during starvation, when i t is living on its body-protein stores and is temporarily “carnivorous” (144). Moreover ability of rats to methylate nicotinamide is known to vary with the straiii, such variations being genetically inherited (223). Birds, e.g., the chick (181), can conjugate nicotinic acid with ornithine. The compounds principally encountered in the animal kingdom are illustrated in diagram 22.

Identification of N-methylnicotinamide as an important nicotinic metabolite is due to Huff and Perlzweig (418,429), and the 6-pyridone was identified by Knox and Grossmann (487, 488). Care should be taken over no- menclature. The naturally occurring metabolite (diagram 22) may be referred to as N-methyl-6-pyridone-3-carbonamide or (more correctly from the chemical point of view) as N-methyl-2-pyridoned-carbonamide. Chem- ical oxidation of N-methylnicotinamide can give both possible a-pyridones (683), that illustrated in diagram 22 and its isomer with the keto group between nitrogen and carbonamide groups. The latter is systematically named N-methyl-2-pyridone-3-carbonamide. It is therefore probably

For more extensive reviews see references 493 and 812a.

METABOLISM OF THE AROMATIC AMINO ACIDS 103

better, to avoid confusion, to refer to the natural metabolite as the 6-pyridone.

Nicotinic acid can be amidated fairly readily in mammals, but hydrolysis of the amide occurs to a lesser extent. In microorganisms (e.g., 655) and insects (e.g., 843) deamidation appears to occur more readily. Nico- tinic acid is also probably more easily broken down by microorganisms, this degradation occurring via 6-hydroxynicotinic acid (420).

Nicotinic acid is also excreted as the glucuronide by the rat (249a). The conversion of N-methylnicotinamide to its 6-pyridone has been

studied enzymically (429). The isomeric 2-pyridone may well be formed in small amount (542)) but most methods for determination do not distinguish between the isomers (e.g., 397). The 6-pyridone is not hydrolyzed to the corresponding acid (398; but see 1012). The enzyme of rat liver converting nicotinic acid to nicotinuric acid occurs in the mitochondria (454). Methylation of nicotinamide is carried out by a soluble enzyme, nicotinamide methylkinase, which has been studied by Cantoni (135, 136). The methyl group is derived from methionine.

The fate of administered nicotinic acid and nicotinaniide has been studied in many species including man (696), the rabbit (143), rat (426, 542, 561, 696)) and mouse (542, 738) with the use of isotopic and other techniques. The results agree in showing that the amount of nicotinic acid or nicotinamide degraded completely to carbon dioxide is comparatively small ; the greater part is excreted as a mixture of the metabolites shown in diagram 22, the proportions of the mixture varying with the species and the dose level. These results imply that there is no readily available route in higher organisms from nicotinic acid and its simple derivatives to aliphatic compounds, and they therefore raise the question of how tryptophan, which is generally supposed normally to be degraded via nicotinic acid, is ultimately metabolized.

A possible route for the latter is suggested by irradiation experiments on pyridine nucleotides, using both ultraviolet (797) and X-irradiation (831). Such experiments may give results analogous to those normally occurring in biological oxidations. It was found that splitting of the pyridine ring could occur, and also reduction to unidentified products. Such products if formed naturally might be degraded to aliphatic compounds. Is it therefore through the pyridine nucleotides, rather than through nicotinic acid, or even through an earlier precursor of nicotinic acid and the pyridine nucleotides, that the main stream of tryptophan metabolism flows?

1’111. ‘rRYPTOPHAN DEGH.\DATION B Y T H E E N T E R A M l N E -

SEROTONIN PATIIWAY

As a result of extensive study of a system of cells, which he called the criterochromaffin system, occurring particularly in the skin and gastroin-

104 C. E. DALGLIESH

testinal mucosa of vertebrates, Erspamer deduced that it must contain a physiologically highly potent material to which in 1933 (892) he gave the name enteramine. Particularly rich sources of enteramine were found in the skin of amphibia and in the salivary glands of the octopus. Erspamer (review, 237) made detailed chemical (893) and pharmacological (230-232) studies which enabled him by 1948 (234) to characterize enteramine as an indole derivative also carrying one or more phenolic groups and a side chain having a terminal primary or secondary amino group.

Meanwhile Rapport and his colleagues had been investigating the vasoconstrictor substance in serum which they named serotonin, and isolated in 1948 (693,694). The isolated material was shown (692) to be a complex of creatinine with what was thought to be 5-hydroxytryptamine (structure diagram 23) and identification of the latter was confirmed by synthesis (19, 341, 363, 821). It then became clear that enteramine and serotonin were identical (e.g., 238, 239), and an enormous amount of work on the physiology and pharmacology of 5-hydroxytryptamine has subsequently appeared (reviews 235a, 237, 660, 958).

5-Hydroxytryptamine is a substance of great physiological potency and is agreed to be probably a hormone as important in normal physiological processes as are adrenaline, noradrenaline, acetylcholine, and histamine. Even so, its exact significance is not yet clear. It has a profound effect on smooth muscle (e.g., 699 and reviews above) and hence influences blood vessels and blood pressure; its vasoconstrictor properties were of course the basis of Rapport’s isolation. Gaddum (282, 283) has shown that specific receptors exist, called by him “tryptamine receptors,” which are insensitive to adrenaline, acetylcholine, and histamine, and very sensitive to hydroxytryptamine. Hydroxytryptamine also occurs in the central nervous system, and Woolley and Shaw (958, 959) consider it to play a vital part in mental proceses. They attribute various mental disorders, such as certain forms of schizophrenia, to inadequate production of hydroxytryptamine in the brain. On the other hand, in various forms of cancer an excessive production of hydroxytryptamine appears to occur in the tumor (544, 545). This can be reflected in the symptoms (e.g., 870) or in an excessive urinary excretion of its degradation product, 5-hydroxyindoleacetic acid (149,389). Erspamer (236) considers the prime role of enteramine to be that of a hormone controlling renal function.

5-Hydroxytryptamine is also an active constituent of many venoms, e.g., wasp venoni (441) and toad venom (237,883).

1. Biospthesis of 6-Hydroxytryptamine

It seemed likely that enteramine was formed either froni tryptophan or If tryptophan were the precursor two biosynthetic possibly from tyrosine.

105 METABOLISM OF THE AROMATIC AMINO ACIDS

pathways were possible; tryptophan -+ tryptamine -+ 5-hydroxytryptamine, or tryptophan -+ 5-hydroxytryptophan -+ 5-hydroxytryptamine, If tyrosine were the precursor this might be converted to 2,5-dihydroxyphenylalanine and then to 5-hydroxyindole1 a cyclization in vitro (165, 360), followed by addition of the side chain:

'CH.COzH .-a \

H HoacHz OH NHz '

occurring readily

-- etc.

2,5-Dihydroxyphenylalanine 5-K ydroxy indole

Elucidation of the actual biosynthetic route in mammals as that shown in diagram 23 is due to Udenfriend and his colleagues. Biogenesis from tyrosine almost certainly does not occur in the animal organism (in which 2,5-dihydroxyphenylalanine is not a primary tyrosine metabolite) but may well occur in plants and may play a part in biogenesis of certain alkaloids.

Udenfriend and his colleagues (885) found in animal kidney extracts an enzyme which specifically decarboxylated 5-hydroxytryptophan (217, 218) to 5-hydroxytryptamine, but was without action on tryptophan, 7-hydroxy-

Tryptophan

5-hydrosy tryptophan 5-hydroxytryptophan decarboxylaae I

amine H o ~ - - ~ ~ 2 - ~ ~ 2 ~ ~ ~ z

~ ~ ~ C - O ] H - H ~-IiydIoa)-indolcncetaldehyde 5-hydroxytryptamine

(enteramine, serotonin) I

5-liydiosyindoleacet ic :xiti methylated conjugated der ivat ivcs derivatives

(Diagrani 24)

Pathway for mammalian synthesis and breakdown of 5-hydroxy- Diagram 23. tryptamine.

106 C. E. DALGLIESH

tryptophan, or tyrosine. This obviously suggested that 5-hydroxytryptophan, rather than tryptamine, was the intermediate in hydroxytryptamine biosynthesis. Moreover 5-hydroxytryptophan, 5-hydroxytryptamine, and N-methylated derivatives (discussed later) occur together in the venom of the tropical toad (883). On giving (2-C'*)-~~-tryptophan to the toad the hydroxytryptamines became radioactive. Moreover radiocative 5-hy- dr~xy-C~~-tryptophan (isolated after addition of carrier) was formed from the labled tryptophan by liver slices (884), conclusively demonstrating the the pathway for enteramine bi osynthesis .

5-Hydroxytryptophan was not metabolized by a tryptophan-adapted strain of Pseudomonas (217) and was not attacked by the tryptophan peroxidase-oxidase system (217, 884). The enteramine and kynurenine pathways are quite distinct, as is supported by the facts that synthetic 5-hydroxykynurenine (124, 574), the expected product of tryptophan peroxidase-oxidase action, does not act as an ommochrome precursor in insects or as a nicotinic acid precursor in Neurospora (124).

No details have yet been reported of the system converting tryptophaii to 5-hydroxytryptophan. However 5-hydroxytryptophan decarboxylase has been studied in some detail (148). It occurs in the kidneys and livers of all animals tested; in the guinea pig the stomach contains more than the liver, and it also occurs in the lung. The enzyme is inhibited by carbonyl reagents (148)) and though this inhibition is not reversed by pyridoxal phosphate, the enzyme is considered probably to be Bs-dependent, as are other amino acid decarboxylases. It is also inhibited by chelating agents (49), and this inhibition is reversed especially by Mn* and Mg+. Dialysis does not remove the coenzyme. The enzyme is highly specific, attacking L-, but not D-, 5-hydroxytryptophan, and is quite distinct from dopa decarboxylase. The amounts of the enzyme and its wide distribution empha- size the importance of the pathway.

The circulating hydroxytryptamine in the blood occurs solely it1 the platelets (424, 889,978), provided no platelet damage has occurred allowing its release into the serum. On the other hand, no 5-hydroxytryptophaii (977) or, in general, hydroxytryptophan decarboxylase occurs in platelets, and they are therefore unlikely to be in the normal site of hydroxytryptamine biosynthesis. Humphrey and Toh (425) have shown that 5-hydroxytryptamine is absorbed by blood platelets and they consider that it is formed in some other orgaii, such as the gastrointestinal tract (872), which is known to contain large quantities of enteramine (179,256)) and is then absorbed by the platelets during circulation. They consider that platelets have the dual function of keeping the blood plasma normally free of hydroxytryptaminr, and also of allowing local release of the vasocoiistrictor from damaged platelets, bringing about hemostasis. On the other hand, Udciifriend and

METABOLISM OF THE AROMATIC AMINO ACIDS 107

colleagues (889) have been able to introduce C1*-hydroxytryptaniine into the rabbit platelet. The half-life of the hydroxytryptamine in the platelet was equal to the half-life of the platelet, and they consider that the normal site of hydroxytryptamine biosynthesis is therefore the same as the normal site of platelet formation, which is generally considered to beeither the bone marrow or the lung (935). But the wide distribution of hydroxytryptophan decarboxylase suggests that such are not the only sites of synthesis. Er- spamer (236) has advanced evidence against a common site for cnteramine biosynthesis and platelet formation.

2. Degradation of 5-Hydroxytryptumine

When 5-hydroxytryptamine is liberated into the plasma rapid degradation occurs. It has been known for a considerable time that tryptamine is rapidly converted to indoleacetic acid both in vivo and in perfused tissue (249, 325). This reaction is brought about by amine oxidase (82, 681, review 77), which equally attacks 5-hydroxytryptamine (76, 81, 278) and can conveniently be thus detected (79). 5-Kydroxytryptamine is readily converted to 5-hydroxyindoleacetic acid by liver and kidney hom*ogenates (871) and the reaction is blocked by semicarbazide, suggesting that the expected 5-hydroxyindoleacetaldehyde (diagram 23) is an intermediate. 5-Hydroxyindoleacetic acid is a normal urinary excretion product, about 10 mg. per day being excreted by man (871). Experiments with isotopically labeled tryptamine in the mouse (767) suggest that amine oxidase is the sole degradative pathway used, and the same may well apply to 5-hydroxytryptamine. A large part of the urinary excretion of indoleacetic acid is in the form of the glycine conjugate, indoleaceturic acid (249, 767). The same may also apply to 5-hydroxyindoleacetic acid (but cf. 23513, 236).

5-Hydroxytryptamine may itself undergo conjugation. Substances closely related to it, e.g., Erspamer’s enteramine I, have been reported in several cases (e.g., 179, and review 237).

N-Met,hylated derivatives may also be formed in mammals (113a).

3. N-Meth ylated Derivatives of 5-H ydrox ytr yptamine

Wieland and co-workers (913) in 1931 isolated from the skin secretion of the toad two indole derivatives, bufotenin and bufotenidin, whose correct structure (diagram 24) was established soon after (914) and confirmed by synthesis (416, cf. 363). Dehydrobufotenin was isolated in 1935 (443) and the previously isolated bufothionin (915) shown to be its sulfate ester (916, cf. diagram 24).

These substances are fairly widely distributed amongst amphibia (197, 444, review 196) and have a powerful physiological action (e.g., 233,235). Their occurrence and pharmacology have been extensively studied by Er-

108 C. E. DALGLIESH

Tryptophan I

1 5-hydroxy tryptophan

5-h ydroxy tryptaniinc (enteramine)

Hoa~ CHn.CI12* ;Me*

H Bufot enidin

OH-

H o o 7 ~ ~ 2 s ~ ~ , v ~ ~ ~ e Hoa7 CH, C H ~ . N M ~ ~

€1 .H N-Methylenteramine Bufotenin

Bufothionin Dehydrobufotenin

Diagram 24. Methylation products of enteramine (6-hydroxytryptamine) occurring especially in amphibia.

spamer (e.g., 237, 240, 242). The coexistence of 5-hydroxytryptophan, 5-hydroxytryptamine, and N-methylated derivatives (883) together with the frequent co-occurrence of related derivatives of tyrosine, suggests the biosynthetic sequences shown in diagram 24 (cf. 242).

Bufotenin also occurs (919) in Amanita mappa, a poisonous fungus species related to Amanita phalloides, which produces phalloidin. Such organisms can obviously carry out varied types of tryptophan metabolism.

IX. ROUTES FOR TRYPTOPHAN DEGRADATION USED PRINCIPALLY BY MICROORGANISMS

The work already described has shown that a large number of microorganisms degrade tryptophan partly or entirely by the rovte used in higher organisms. Stanier and his co-workers (826-828, 830), using the method of simultaneous adaptation, examined many strains of Pseudomonas and found that all degraded tryptophan to kynurenine. The majority of the strains then split kynurenine to anthranilic acid, which was converted to catechol and the latter degraded as already described (p. 78). This they called the “aromatic pathway.” A few strains converted kynurenine to kynurenic acid, which was further degraded by a route not as yet established. This they called the “quinoline pathway.” One strain could use both routes (874).

METABOLISM OF T H E AROMATIC AMINO ACIDS 109

anthranilic acid --t catechol --* etc.

P aromatic ptlthww /

\ ;l;ylog \

Kynurenine

I kynurenic acid - unidentified products

The bacterial enzymes involved in these changes, where known, have already been described.

Besides using such specific pathways, bacteria can degrade tryptophan by many of the more general pathways applicable to other amino acids. Such pathways in the case of phenylalanine and tyrosine have been given in diagram 16 (p. 77), and similar pathways apply to tryptophan (putting Ar = indol-3-yl in this diagram).

The existence of tryptophan decarboxylase has been claimed by many workers (e.g., 248). Gale and co-workers (cf. 284) failed to find it, but this might be due to enzyme lability. It is probable that if tryptamirie were formed a t least some orgariistns could degrade i t by amine oxidase to indoleacetic acid.

Tryptophan can be converted to indolepyruvic acid either by oxidative deamination or by transamination (e.g., 739, 912) and the indolepyruvic acid can give rise to indoleacetic acid. The fate of indoleacetic acid formed by the bacterial flora of the mammalian gut is discussed below. Bacterial indolelactic acid (e.g., 757) is presumably derived from indolepyruvic acid, but indolelactic acid excreted by mammals (e.g. 17) may be of true mammalian rather than bacterial origin. Indolepropionic acid can also be formed by bacteria (e.g., 412, 633), but further metabolism in mammals of any indolepropionic acid formed in the gut is still obscure (904). Skatole (3- methylindole) has long been known as a product of bacterial decomposition of protein and is formed from tryptophan not only by the bacterial flora of the gut but also in putrefying secretions, e.g., sputum (756). It may well arise by decarboxylation of indoleacetic acid.

1. Urinary Indoleacetic Acid and Indoleaceturic Acid. Urorosein

Nencki and Sieber (634) in 1882 described a beautiful red color obtained on adding concentrated hydrochloric acid to certain pathological urines. Much confusion exists in the older literature between this Nencki-Sieber reaction, the Salkowski reaction (417, 745) (a red color with hydrochloric acid and dilute ferric chloride), and the urorosein reaction (a red color with hydrochloric acid and nitrite), but the same chromogen is probably mainly

110 C. E. DALGLIESH

responsible for all. Rosin (736,737) considered the chromogen to occur to some extent in all urines, but not all workers agreed with this (e.g., 287) The reaction was studied especially by Herter (390), who isolated the chromogen and identified it as indoleacetic acid. Later work (249,409) has show1 that in fresh urine the principal chromogen is iiidoleaceturic acid (indol-3- ylacetylglycine). The colored compound formed has been isolated (“22) and shown to be a diindolylmetheiic (362). 111 the author’s experience indoleacetic acid and indoleaceturic acid are both normal human uriiiary constituents (cf. also 401, 659), the latter predominating. Indoleacetic acid is the sole plant auxin occurring in urine (918).

It is probable that much of the indoleacetic acid is derived from bacterial degradation of tryptophan in the gut, and is then absorbed into the blood stream and to a large extent conjugated with glyciiie in the liver. Thus excretion can be increased in presence of an abnormal intestinal flora (390). On the other hand, a t least part may arise from plant material in the diet (330, and cj. subsequent discussion of heteroauxin). Moreover the possible formation of indoleacetic acid from tryptophan in animal tissues deserves investigation. Indoleacetic acid has a marked effect on amino acid trans- port into cells (147, 704) and may conceivably have a normal physiological function. The suggestion (494) that indoleacetic acid affects a mammalian growth has not been substantiated (517, 733, 753).

2. Bacterial Degradation via Indole. The Tryptophanase fieaction

The formation of indole during bacterial putrefaction of protein-containing materials has been known for a considerble time (e.g., 632, 746). Hopkins and Cole (412), soon after their discovery of tryptophan, investigated the action on it of Escherichia coli. They found that aerobic degradation of tryptophan gave indole and indoleacetic acid (cf. 746), whereas anaerobic degradation gave indolepropionic acid. The formation of indole was, and still is, widely used for differentiation of bacteria, but the biochemistry of indole formation was somewhat neglected. It was generally Considered that indole was formed by way of a number of derivatives wit,h successively shorter chains, eg., indoleacrylic acid, indolepropionic acid, ethylindole, indoleacetic acid, skatole, and indolecarboxylic acid. The direct formation of indole by cleavage of the tryptophan side chaiu, without participation of any indolic intermediate, became clear from the work of Woods (954) and Happold and Hoyle (347), and the enzyme bringing this about was named tryptophanase (347). Considerable work has been carried out on the structural requirements of the tryptophanase substrate (e.g., 28,46). The reaction is catalyzed by its products, indole and pyruvic acid (46). Besides pyridoxal phosphate, discussed below, potassium or ammonium ions (348) are required and possibly also iron (194).

METABOLISM OF THE AROMATIC AMINO ACIDS 111

Numerous hypotheses, subsequently proved incorrect, have been suggested for the mechanism of tryptophanase action (see review by Happold, 345, covering the subject up to 1950). With the demonstration, already discussed under biosynthesis, that tryptophan is formed from indole and serine, i t was thought possible that tryptophanase carried out the reverse of this reaction. However the synthesizing enzyme (890) was distinct from tryptophanase (951), though both require pyridoxal phosphate as coenzyme. Tryptophanase gives rise (951) to equimolar amounts of indole, pyruvic acid, and ammonia, but riot to serine, and no oxygen uptake is involved. Neither alanine nor serine is deaminated, and these cannot therefore be intermediates. The mechanism of function of pyridoxal phosphate in the reaction has already been discussed (p. 91). It has been concluded (300) that in the enzyme-coenzyme-substrate complex, L-tryptophan is bound to the enzyme through the pyrrole nitrogen and the side chain carboxyl, the a-amino group is combined with the carbonyl group of pyridoxal phosphate, and the phosphate group binds coenzyme and apoenzyme. Ex- periments with atomic models are consistent with this interpretation (27).

The adaptive formation of tryptophanase has been studied by Happold and his colleagues (202, 339, 340).

3. Further Degradation of Indole by Bacteria

Some bacteria can further degrade indole. This reaction has been stud- Experiments using successive adaptation ied by Uchida and colleagues.

(742) suggested the following pathway:

Indole Isatin Formylanthranilic acid

etc. Catechol c-- Salicylic acid - Anthranilic acid

Ring-opening to give formylanthranilic acid is analogous to the formation of formylkynurenine from tryptophan. Enzymic experiments on the ring- opening step (877) led to the conclusion that rupture of the C-C bond involved an oxidase in which vitamin B I ~ is possibly concerned as coenzyme; this is thought to be followed by a dehydrogenase connected with the pyridine nucleotides, and possibly linking up with the folic acid system.

4. Origin of Urinary Indican, Indigo, Indirubin, Skatoxyl, and Skatole Red

Urinary indican is the 0-sulfate of indoxyl (usually isolated as the potassium salt, 414) and is excreted by mammals as a detoxication product of the

112 C. E. DALGLIESW

indole formed from tryptophan by the bacterial flora of the gut. The horse excretes particularly large amounts (364) The indole is absorbed through the gut wall into the blood stream. Part is detoxicated in the gut wall (647), the rest is detoxicated mostly in the liver, but to some extent in other organs (835). Some indoxyl may also be conjugated as the 0-glucuronide (639).

glucuronide / - O - - O H QQ

Indole H - H H

Indican

ace, ,c=c <NHyJ \

N H co

Indigo

Q aC0 ,C=C \ NH

NH 'c' 0

Indirubin

Indican was early considered to be a normal constituent of human urine (413), but this was disputed by later workers, probably owing to the inadequate sensitivity of the tests employed. Its normal occurrence is now established (87,175). Indole produces nausea, headache, and other unpieas- ant symptoms (649), whereas indican is pharmacologically almost inactive (435). Indican formation is therefore a true detoxication, but in man only about 30 % to 50 % of ingested indole is converted to indican (87,649). A good deal of the indole produced in the gut appears in the feces, indole and the related skatole (3-methylindole) being largely responsible for the typical fecal smell.

Indican excretion tends to rise when there is an increase in protein intake, or an increase in endogenous protein breakdown as in fever, or in constipa- tion or other intestinal blockage owing to the increased opportunity for bacterial action. Though there is little doubt that a bacterial origin is to be attributed to a great part of normal indican and indole excretion, there is evidence suggesting that there may be other sources. This point was early debated (e.g., 85, 219). Homer (409) suggested that indican could arise from indolealdehyde formed by a disturbed metabolism of tryptophan in the liver. But indolealdehyde is not indoxylogenic if the digestive tract is removed (835). On the other hand, removal of indole-forming organisms

METABOLISM O F THE AROMATIC AMINO ACIDS 113

from the gut by intensive chemotherapy does not necessarily prevent, of even diminish, indican excretion (155, 956). It remains possible that indole could arise in some mammalian tissue.

Chromogens related to indican may be precursors of the indigo and di- bromoindigo (“Tyrian purple”) formed in some marine organisms (100a).

Hydrolysis of indican gives indoxyl, which is readily oxidized to indigo and indirubin, as shown in the diagram above. “Indigo stones,” though rare, have been known for a considerable time (e.g., 146, 656), and these could be formed from indoxyl which has not been conjugated with sulfate, or from indican, which is readily hydrolyzed under acid conditions and may also be hydrolyzed by arylsulfatases. Indirubin is encountered more fre- quently in urine (cf. 618), and though its occurrence has received detailed study (705,736), it is not clear whether i t is formed in the body and excreted as such or is merely derived from urinary indican after excretion

Skatole also arises by action of the intestinal flora and was early claimed (105) to give rise to a substance “skatoxyl,” supposedly analogous to indoxyl, and to “skatole red.” Homer (409) produced suggestive evidence that “skatole red” is probably a mixture of indigo and indirubin The formulation of skatoxyl is inherently improbable chemically, and unpublished preliminary experiments of the author suggest that its postulated existence will not withstand modern methods of investigation.

X. TRYPTOPHAN METABOLISM IN PLANTS. HETEROAUXIN Tryptophan can be metabolized in at least some plants by the kynurenine

or closely analogous pathways. For example, tryptophan gives nicotinic acid in many green plants (329) ; both tryptophan and hydroxyanthranilic acid are converted to nicotinic acid in maize (626, 627) and kynurenine is also known in plants (e.g., 521). A mutant of maize obtained in the Bikini experiments accumulates large quantities of anthranilic acid (862). Tryp- tophan appears to be converted to nicotinic acid in germinating green gram (PhaseoZus mungo) by a pathway similar to that in animals (798, 799). Wiltshire (932) in a detailed enzymic study of tryptophan oxidation in pea seedlings concluded that the greater part of the tryptophan was metabolized by a peroxidase system showing marked analogies to the Knox-Mehler system in animals, and the metabolism probably proceeded through kynurenine and hydroxykynurenine. The rate of degradation of tryptophan by this pathway was more than 100 times as fast as the rate of conversion of tryptophan to indoleacetic acid reported in tobacco (922).

A remarkable advance in plant physiology occurred when Kogl and co- workers (496) isolated a plant growth hormone, called heteroauxin, from human urine. This was identified as indole-3-acetic acid and was shown (497, 498) also to occur in plants, and in yeast from which i t was isolated.

114 C. E. DALGLIESH

The work of many investigators soon showed i t to be extremely widespread in plants, and it became clear that it was a fundamental plant hormone (e.g., reviews 530, 655a). Excessive production of indoleacetic acid by parasites is responsible for certain types of plant tumor (e.g., 948). The synthetic plant growth regulators, which are in general substituted phenoxyacetic. acids, probably function as indoleacetic acid analogues.

1. Biogenesis and Degradation of Indoleacetic Acid in Plants

Tryptophan has been clearly established as the precursor of indoleacetic acids in both plants (e.g., 303, 921, 922) and fungi (e.g., 864), and in plant tumor tissue (e.g., 378, 948). Two routes are possible for indoleacetic acid formation from tryptophan as follows:

amine oxiduse decar boxyla tion

1 Tryptophan P tryptamine

I dpamination or transamination 1 indoleacetaldehyde

-1 indolepyruvic acid - * indoleacetic acid

indoleaoetic acid oxidaso I unknown products

There is evidence that both these routes can occur. The enzymes coil- verting tryptophm to indoleacetic acid can be obtained in maize embryo juice; the tryptophaii is thought to arise from the endosperni (964). In- dolepyruvic acid is also present in maize endosperm (837, 838), suggesting it to be an intermediate. On the other hand, tryptamine is converted to indoleacetic acid in plants (304, 815) and the amine oxidase responsible has been studied by Kenten and Mann (464). Consideration of the biogenesis of alkaloids, discussed later, suggests that both tryptainine and indoleacetaldehyde are likely to occur in plants.

Indoleacetic acid is degraded in plants by a specific iiidoleacetic acid osi- dase. This is a light-activatable flavoprotein enzyme coupled through hydrogen peroxide to a peroxidase (285; but cf. 463a, 805b). It apparently uses phenols as cofactors (296) but can be inhibited by polyphenols (305). The product of the reaction is still unidentified (836).

2. Other Indolic Plant Growth Hormones

An auxin in apple endosperm (5G5) not identical with indoleacetic acid ivas identified as ethyl indoleacetate (863, cj. 697), but this may be an arti- fact due to esterification during isolation (377).

METABOLISM OF THE AROMATIC AMINO ACIDS 115

Other indolic auxins besides indoleacetic acid occur in plants (e.g., 396). Both indoleacetaldehyde (51, 107) and indoleacetonitrile (51, 377, 452) have been claimed to be plant growth hormones. Indoleacetonitrile has heen isolated from plant sources (377) and shown to be of wide distribution, but whether it is a hormone per se or only acts as a precursor of indoleacetic acid is not yet clear (cf. 838, 865).

A11 these indolic plant hormones or hormone precursors are likely to arise from tryptophan. Other reactions of trytophan, for example, in alkaloid hiogenesis, are discussed in the next section.

XI. N A T U R A L PRODUCTS PROBABLY RELATED TO THE

AROMATIC AMINO ACIDS

In higher organisms the number of products derived from the aromatic amino acids is comparatively small. In lower organisms, such as bacteria and plants, the biosynthetic possibilities are enormously greater. In this section very brief mention will be made of some types of compound probably metabolically related to the aromatic amino acids.

A large proportion of the compounds mentioned will be of plant origin. Plant biochemistry has lagged considerably behind mammalian biochemistry, and relatively much less is known of the metabolic processes occurring, Though it is usual to regard many of the substances discussed as ultimately derived from the aromatic amino acids, it should be emphasized that there is still little direct evidence on this point. The changes postulated in theories of alkaloid biogenesis in general involve biochemically plausible reactions, but the result in some cases could be equally well explained on the assumption that both alkaloids and aromatic amino acids are derived from common precursors, e.g.,

aromatic amino acids

natural products

This applies especially to substances such as the essential oils or lignin. The comparatively small amount of work so far done on the biogenesis of alkaloids from isotopically labeled amino acids suggests that theories of hiogenesis of alkaloids from amino acids are probably justified.

I. Probably Related Metabolic Products in Microorganisms

Of simpler substances can be mentioned phenylacetic acid as a constituent of benzylpenicillin; O-methyltyrosine in, e.g., the antibiotic puromy-

116 C. R. DALOLIESN

ciii (903) ; the antibiotic. chloramphenicol; the Pyo antibiotiw (2-nlkyl--l- hydro?cyyuiiiolints, 368) and their N-oxides ( I 581, prohably derived froin kynurmic acid.

2. E'rohubly 11'clatcd dlctabolic Products in Plants and Fungi

Simple examples are: p-hydroxybenzylisothiocyanatc in white mustard (472); the substance p-Me0.CBHI.CH2.CH2.NMe.C0 .CH : CT3 .CsH6 i i i

Southern Prickly Ash (526) ; circumin (cf. 822) ; chlorogeiiic and isochloro- genic acids, which are widely distributed glycosidic derivatives of 3,4-dihy- droxycinnamic (caff eic) acid (cf. 432) ; vanillin (3-methoxy-4-hydroxybtllz- aldehyde) and related substances such as many of the essential oils, e.g., anethole, eugeiiol, saff role; gallic acid (3,4,5-trihydroxybenzoic acid) and numerous other phenolic substances from, e.g., tannins; 3 ,4-dihydroxyphenylacetic acid, 2,5-dihydroxyphenyIglyoxylic acid, and many related substances in fungi (e.g., 609,687) ; some fungus pigments such as cortisalin, p-HO.CeH:- (CH:CH),~COIH (312), and violacein which contains both 5- hydroxyindole and oxindole nuclei (43,44) ; sulfur derivatives of oxindolyl- alaiiine (a-hydroxytryptophan) as in phalloidin (1 57,920).

3 . Flavonoids and Liynin

A vast number of flavonoid conipounds ocrur in nature and these may well be derived from the aromatic amino acids. Birch arid co-workers (66) have demonstrated the following hiosyrithetic pathway in the green alga C'hlam- ydomonas agamctos:

Phenylalanine

1 1

tyrosine

3,4-dihydroxyphcnglalanine

:< ,4-dihydroxyphenylpropionic acid iriositol

3,4-dihydroxycinnarnic acid + phloroglucinol ----A

L--T precursor X

1 quercetin, etc.

Lignin, which represents some 20 % to 30 % of wood, can be regarded a h

a polymer formed from phenylpropane skeletons, such as 3-methoxy-4-hy-

METABOLISM OF T H E AROMATIC AMINO ACIDS 117

droxycinnaniic acid (e.g., 740). propane skeletons of phenylalanine, tyrosine, and dopa (cf. 107a).

This suggests a close relation to the phenyl-

4. Alkaloids

The relation of many of the simpler alkaloids to the aromatic amino acids is obvious. For example, germinating barley contains (241) , besides tyrosine and tyramine, N-methyltyramine, NN-dimethyltyramine (hordenine), and the trimethylammonium derivative (candicine). In this simple case the N-methylated derivatives are known to be derivable from isotopically labeled tyraniine (538) and the methyl groups are known to arise from methionine by transmethylation (540, 586). Similarly N-methyl derivatives of phenylethylamine, 3 , 4-dihydroxyphenylethylainine, and 3 , 4,5-trihy- droxyphenylethylamine are well known alkaloids (cf. review, 701). N- Methylated derivatives of tryptamine and hydroxytryptamine equally occur; for example, eserine has an obvious relation to 5-hydroxytryptamine. Methylated derivatives of metabolites of the aromatic amino acids also occur, for example, trigonelline (67), which is the betaine of nicotinic acid, and damascenine is probably similarly related to hydroxyanthranilic acid.

Eserinc Cirainine

The comparatively simple alkaloid gramiiie is particularly interesting, as the nitrogen is here separated from the indole nucleus by only one carbon atom. Almost all other indole alkaloids contain the aminoethyl side chain found in tryptamine. If some alkaloids were formed from precursors of the aromatic amino acids rather than from the amino acids themselves, one would expect gramine to fall into this category. Yet isotopic experiments have made it clear that the indole nucleus and the side chain CH2 group of gramine arise from the indole nucleus and side chain &carbon, respectively, of tryptophan (101, 539). By analogy alkaloidal derivatives based on benzylamine are probably derived from phenylalanine.

Theories of the biogenesis of more complex alkaloids are due to Pictet (Wig), Winterstein and Trier (934), and particularly Robinson (707, and later discussions, 708, 709, 711, 712), and a vast amount of work has been done on syntheses under “physiological conditions,” i.e., under conditions and with the use of substances likely to occur in the plant (for reviews and discussions see, e .g . , 422,577,578,775 and for reviews on more plant physiological aspects of alkaloids see 195, 440).

Winterstein and Trier pointed out that benaylisoquinoliiie alkaloids

118 C. E. DALGLIESH

Me0

Me0 +

M e o q N H Me0

Dimethoxyptienylethylamine and dimethoxyp henylacetaldehyde

Te trahydropapaverine

Diagram 25. The type of reaction possibly involved in benzylisoquiIioline alkaloid biosynthesis.

could be formed from two iiiolecules of dibydroxyplienylalaiiine. To tahr a specific iristance (819) 3,4-diinethoxyphenylethylamirie condenses with 3,4-diniethoxyphenylacetaldehyde under “physiological miditions” to give tetrahydropapaverine (diagram 25) . Amine oxidase occurs in plants (e.g., 464, 909), and if it were to act on the above amine, both aldehyde stud amine would be present together, and conditions for tetrahydropapaverine formation would therefore be very favorable. Similarly acetaldehyde with, say, tyramine would give a much simpler isoquirioline derivative (776).

It may not, however be necessary for decarboxylatiori of the amino acid to occur. A condensation with the amino acid similar to that above \vould give the same result if decarboxylation occurred after condensatiori. Simi- larly an aldehyde coinponerit need not be present as such; a potential aldehyde, such as a keto or imino acid, also condenses readily (c.g., 334,337). Again the same product is obtained by subsequent decarboxylation. Transaniination is known to occur in plants (e.g., 931) and the occurrence of keto acids is to be expected. Both amino slid keto acids could of course be formed from the amino acid by oxidative deamination.

The comparatively complicated molecule of emetiiir (diagram 26) can be derived (710) from three molecules of dihydroxyphenylalaiiiiie by making use of the Woodward (955) hypothesis discussed below. In this, as in iiiany other cases, biogenetic considerations have played a large part in dtter- milling the correct structure, and biogenetic theories thereby gain strong albeit indirect, support.

The indole alkaloids provide an even richer source of biogenetic interrelationships. Thus, condensation of tryptamine and dihydroxyphenyl- acetadehyde (or equivalent precursors) under conditions similar to those already described gives a tetrahydro-harman derivative (diagram 26; cf. 336,338). Further condensation of this with formaldehyde (cf. 335) (which may be biogerietidly derived from, say, serine or glycine) gives the same basic skeleton as in the alkaloid yohimbine.

METABOLISM O F THE AROMATIC AMINO ACIDS

OMe OMe

E t Emetine

119

H Tryptaminc

+ CHO

OH Dihy droxy phen y lacetaldeh yde

oxql+* CHnO

/ /

' OH ' OH

Compound with yohimbine A benzyltetrahydroharman skeleton derivative

Emetine; and biosyrlthesis of yohimbine-type alkaloids.

OH OH

Diagram 26.

In yohimbine formation the initial Mannich-type condensation of the aldehyde with the tryptamine was assumed to occur in the a-position of the indole ring. Similar condensation in the ,&position (which already carries the ethylamine side chain) can give an intermediate which Wood- ward (955) pointed out can give rise to the very complicated molecule of strychnine. The stages, shown in diagram 27, involve a ring scission at the catechol grouping and a rearrangement. Similar ring scission is probably also involved in the biosynthesis of emetiiie (diagram 26) and of the quinine alkaloids. The derivation of two of the latter is show in diagram 28 (cf. 308).

The starting product in diagram 28 can be derived from tryptophan, dihydroxyphenylalanine, and formaldehyde in ways obvious from diagram 26. Scission of the catechol grouping can give a dialdehyde compound, in which one aldehyde group can react with the secondary aliphatic amino group (dotted arrow 0) to give, ultimately, cinchonamine. If there is a further scission of the pyrrole ring (dotted line aa) and a rearrangement, cinchonine results.

It will be seen from this very brief treatment that the essential step in

120 C. E. DALGLIESH

&CH-I!JH Tryptamine +

dihydroxyphenyl acetaldehyde

(ct. diagram 26)

Strychnine

several stages

' OH Diagram 27. Woodward's hypothesis for biosynthesis of strychnine from, ulti-

mately, tryptophan and dihydroxyphenylalanine.

CHz * CHzOH CHz. CHnOH

in stages

CH2 * CHzOH

CHa CH * CH : CHZ

OH'

Cinchonine Cinchonamine Diagram 28. Derivation of two typical quinine alkaloids from, utlimately, trypto-

phan and dihydroxyphenylalanine.

the biosynthesis of many alkaloids is a Mannich-type reaction between an active position on an aromatic ring, an aldehyde (which may also be derived from an aromatic amino acid), and the amino group of the original amino acid side chain. Other changes are fairly simple and plausible, such as the introduction of one-carbon and two-carbon units. Besides these reactions

METABOLISM OF THE AROMATIC AMINO ACIDS 121

the various members of the alkaloidal families may differ by the number of phenolic groups and the extent to which 0- and N-methylation has occurred, Isotopic experiments have shown that both 0- and N-methyl groups arise hy transmethylation reactions (132, 204, 540, 586, 786) analogous to those in the animal, and this is in accord with the virtual absence of 0- and N-alkyl substituents other than methyl (or the related methylenedioxy group).

The biogenesis of alkaloids from the aromatic amino acids forms an enormously rich field for work with modern isotopic and chromatographic Cech- niques, which as yet has hardly been touched.

XII. FUTURE PROBLEMS Phenylalanine and tyrosine are degraded in large degree to aliphatic sub-

stances, acetoacetic and fumaric acids, which are metabolically highly active, and there is no difficulty in accounting for the amounts of these amino acids metabolized. Of the various transformations involved, the conversion of p-hydroxyphenylpyruvic acid to hom*ogentisic acid, in particular, needs further investigation. Our inadequate knowledge of this reaction is a reflection of two major biochemical problems: the nature of aromatic hydroxylation reactions, of which the enzymes have been obtained in very few cases; and the biological functions of ascorbic acid, which appears to participate both specifically and unspecifically in a wide range of transformations. In addition, much is still to be learnt of the adrenaline and thyroxine pathways.

No such tidy ending can a t present be given to our picture of tryptophan metabolism. In the adult animal in the steady state as much tryptophan must be degraded as is ingested (irrespective of the fraction used in protein biosynthesis, as old protein is being broken down as fast as new protein is being formed). To take man as an example, tryptophan can, so far as is at present known, be metabolized by three main routes: (1) by the nicotinic acid pathway, (2) by the 5-hydroxytryptamine pathway, and (3) by bacterial action in the gut. The 5-hydroxytryptamine pathway gives as principal end product 5-hydroxyindoleacetic acid. Bacterial action gives rise to a number of excretory products, including indican, indoleacetic, indoleaceturic, and indolelactic acids. In addition some tryptophan is excreted unchanged. All these indolic compounds taken together account for only a small part of ingested tryptophan.

The nicotinic acid pathway potentially gives rise to a number of excretion products, largely due to side reactions, such as anthranilic acid, kynurenic acid, and xanthurenic acid, but normally these are excreted in, at most, small amounts. Presumably, therefore, the greater part of ingested tryptophan should give nicotinic acid or simple derivatives. But normal excretion of nicotinic acid and its derivatives is in any case low, and when

1 22 C . E. DALGLIESH

nicotinic acid or likely derivatives are given to an animal a large ftwtioii is excreted in forms with the ring unbroken.

There would therefore seem to be no readily available pathway from niv- otinic acid to simple aliphatic compounds (unless, as already suggested, this occurs via the pyridirie nucleotides), and hy iiiferencc no rcwlily available pathway for degradation of tryptophan to simple aliphatic, compouids. In short, in our present picture of tryptophan metabolism thcre seems to 1 ) ~ considerable discrepaiicy between our knowledge of what goes in aiid what comes out, and a major problem is to resolve this discrepancy. It may he that some at present unsuspected pathway exists (the 5-hydroxytryptaminc pathway was only recently discovered). It may be that some intermediatc in one of the known pathways is metabolized to aliphatic compouiids by route or to a degree at present unsuspected. It may be that degradation of many intermediates occurs, so that there is a “leakage” to aliphatic compounds all along the pathway. It may even be that metabolism in sonw organ other than liver or kidney plays a prominent part.

In additioti many stages in known pathways of tryptophan metabolism require further investigation, in particular, the intermediate lying betweet 1

tryptophan and formylkynurenine, the hydroxylatiori reaction in conversion of kynureiiie to hydroxykyiiurenine, the intermediates in the wiiver- sion of hydroxyaiithranilic acid to nicotinic acid, aiid the site of synthesis and hormonal function of 5-hydroxytryptamine.

And in the plant kingdom, especially, there lies a rich and barely touched field of investigation-the relation between aromatic amino acids and numerous natural products.

XIII. SUMMABY

The reviex covers those aspects of the metabolism of phenylalanine, tyrosine, and tryptophan not concerned with peptide or protein synthesis or breakdown.

Phenylalariiiie arid tryptophan are “essential” amino acids for higher organisms, i.e., they cannot be synthesized by the organism and must, he supplied in the diet. Tyrosiiie is formed from pheiiylalaniiie aiid is tiot essential if the pheiiylalaniiie intake is adequate. The first section of the review covers biosynthesis, which is confined to lower orgariisnis (bacteria, fungi, plants, etc.). Glucose is the precursor of the aromatic ring and is converted to shikimic acid, such intermediates as are known bciiig showti in diagram 1 , Shikimic acid is a common precursor of the aromatic amino acids and of the bacterial growth factors, p-amino- and p-hydroxybenzoic acids. Such intermediates as are yet known in the final stages of aromatic amino acid biosynthesis are showii in diagrams 2 and 3. Much isotopic

METABOLISM OF THE AROMATIC AMINO ACIDS 123

work has becii done 011 thc conversion of glucose to shikiniic acid, but the evideiice is stJill inadequate to defiiie the pathway in detail.

In higher organisms pheiiylalanine is normally transformed to tryosine, a i d then lirokeii down by the route, p-hydroxyphenylpyruvic acid, 2 , 5 - dihydr'oxypheiiylpy~.uvic acid, hoxnogeritisic acid, rnaleylacetoacetic acid, fumarylacetoaretic acid, aiid finally fumaric and acetoacetic acids (diagram 8). The elucidation of this pathway is described scmihistorically; first are considered in vivo rsperimeiits aiid experiments i i i those rare metabolic disorders (c.g., phenylkctoiiuria and alkaptonuria; diagram 6) associated with inhibition of relevant normal degradativc reactions. Then in witro reactions arc coilsidered, followed by individual consideration of the various steps taken in turii.

Phenylalaniiie and tyrosiiie are also metabolized in higher organisms by two routes which are quantitatively less important but physiologically of the highest importance. The first leads to the adrenal hormones adrenaline (epinephrine) arid rioradrerialirie (norepinephrine) ,which may be formed as in diagram 11; this pathway also leads to melanin (diagram 12). The second leads to the thyroid hormones thyroxine and triiodothyronine, the synthesis and breakdown of which are also discussed.

Tryptophan is metabolized in a wide range of organisms by a pathway involving: an unkno~vn intermediate, forinylkyriureiiiiie, kynurenine, hy- droxykynureiiinc (or its phosphate), hydroxyanthrariilic acid (or its phosphate), two unknown intermediates, and nicotinic acid (diagrams 17,18,21). The pathway is considered both in outline and in detail by individual steps. The relation of other vitamins to the pathway (diagram 19), side reactions giving such substances as aiithrariilic acid, kynureriic acid, and xanthurenic acid (diagram 20) and the further degradation of nicotinic acid (diagram 22) and its relation to the pyridine nueleotides are also considered.

Some tryptophan is also degraded to 5-hydroxytryptamine (enteramine, serotonin; diagram 23) and some by iiitestiiial bacteria, using many pathways. These, a d pathways involved in bacterial degradation of phenylal- aiiine and tyrosiiie are all considered (diagram l G ) , as are the chromogens excreted by man as a result of bacterial action, and such minor pathways as those concerned with hardening of the insect cuticle and formation of insect eye-pigments.

Tryptophan also gives rise to the important plant hormone, indoleacetic acid, and microorganisms and especially plants metabolize the aromatic amino acids to a wide range of natural products, for example, certain anti- biotics, alkaloids (e.g., diagrams 25-28), flavonoids, and possibly lignin. These are briefly considered.

The final section mentions various problems likely to be the basis of future work.

124 C. E. DALGLIESH

ACKNOWLEDGMENTS I thank Prof. A. Neuberger for his stimulating and valuable criticism,

and Dr. B. D. Davis for his consideration of the section on biosynthesis.

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