THE CATABOLISM OF PHENYLALANINE, TYROSINE · THE CATABOLISM OF PHENYLALANINE, TYROSINE AND OF THEIR...

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THE CATABOLISM OF PHENYLALANINE, TYROSINE AND OF THEIR DERIVATIVES. BY A. J. WAKEMAN AND H. D. DAKIN. (From the Herter Laboratory, 819 Madison Ave., New York.) (Received for publication, February 16,1911.) The study of alcaptonuria has yielded results of much value upon which to base speculations as to the mode of catabolism of the aromatic amino-acids, phenylalanine and tyrosine, in the normal and alcaptonuric organisms. As is well known these two amino-acids, both of them important derivatives of proteins, are apparently completely catabolized in the normal organism with production of ammonia or urea, carbon dioxide and water; while in the case of the alcaptonuric both the amino-acids yield homogentisic acid. This latter reaction is very remarkable and involves a rearrangement of the relative positions of the side- chain and hydroxyl groups in tyrosine and also the introduction of a second hydroxyl group. This type of change is associated with the intramolecular rearrangement of substances possessing a quinonoid structure. The inference was natural that a substance of quinonoid struc- ture is the precursor of homogentisic acid.’ Neubauer2 as the result of his extensive investigations has indicated the important part which the cr-ketonic acids play in representing an intermediate stage between the a-amino-acids and the corresponding fatty acids with one less carbon atom. Embder? on the other hand has shown that not only phenylalanine and tyrosine but also homo- gentisic acid yield aceto-acetic acid and acetone on perfusion through an excised normal liver. With these basic facts for guidance Neubauer has constructed the following scheme to represent diagrammatically the catabolism of tyrosine and of phenylalanine in the normal organism. ‘E. Meyer: Deutsch. Arch. f. klin. Med., lxx, p. 447, 1901. 20. Neubauer: Ibid., xc, p. 211, 1909. $Beitr. z. them. Physiol. u. Path., viii, p. 153, 1906. 139 by guest on September 16, 2018 http://www.jbc.org/ Downloaded from

Transcript of THE CATABOLISM OF PHENYLALANINE, TYROSINE · THE CATABOLISM OF PHENYLALANINE, TYROSINE AND OF THEIR...

Page 1: THE CATABOLISM OF PHENYLALANINE, TYROSINE · THE CATABOLISM OF PHENYLALANINE, TYROSINE AND OF THEIR DERIVATIVES. ... acetic acid in such a way that the carbon atom of the carbcxyl

THE CATABOLISM OF PHENYLALANINE, TYROSINE AND OF THEIR DERIVATIVES.

BY A. J. WAKEMAN AND H. D. DAKIN.

(From the Herter Laboratory, 819 Madison Ave., New York.)

(Received for publication, February 16,1911.)

The study of alcaptonuria has yielded results of much value upon which to base speculations as to the mode of catabolism of the aromatic amino-acids, phenylalanine and tyrosine, in the normal and alcaptonuric organisms. As is well known these two amino-acids, both of them important derivatives of proteins, are apparently completely catabolized in the normal organism with production of ammonia or urea, carbon dioxide and water; while in the case of the alcaptonuric both the amino-acids yield homogentisic acid. This latter reaction is very remarkable and involves a rearrangement of the relative positions of the side- chain and hydroxyl groups in tyrosine and also the introduction of a second hydroxyl group. This type of change is associated with the intramolecular rearrangement of substances possessing a quinonoid structure.

The inference was natural that a substance of quinonoid struc- ture is the precursor of homogentisic acid.’ Neubauer2 as the result of his extensive investigations has indicated the important part which the cr-ketonic acids play in representing an intermediate stage between the a-amino-acids and the corresponding fatty acids with one less carbon atom. Embder? on the other hand has shown that not only phenylalanine and tyrosine but also homo- gentisic acid yield aceto-acetic acid and acetone on perfusion through an excised normal liver.

With these basic facts for guidance Neubauer has constructed the following scheme to represent diagrammatically the catabolism of tyrosine and of phenylalanine in the normal organism.

‘E. Meyer: Deutsch. Arch. f. klin. Med., lxx, p. 447, 1901. 20. Neubauer: Ibid., xc, p. 211, 1909. $Beitr. z. them. Physiol. u. Path., viii, p. 153, 1906.

139

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140 Catabolism of Phenylalanine

e;

g W/-\X

O\ /O -

T o= 7-8

< ,/\,- T x

8 8-8

g 8-5 C

t

o- t

c-

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A. J. Wakeman and H. D. Dakin 141

It will be seen from t’his diagram that: (1) Phenylalanine is represented as being converted into

tyrosine or into para-hydroxyphenylpyruvic acid (through the stage of phenylpyruvic acid) by introduction of an hydroxyl group in the para-position.

(2) Para-hydroxyphenlypyruvic acid is represented as being converted into a substance of quinonoid structure which by intra- molecular rearrangement passes over into 2.5-dihydroxyphenyl- pyruvic acid; the latter substance yielding homogentisic acid by oxidation.

(3) The homogentisic acid thus formed undergoes decomposi- tion with formation of “acetone bodies” involving disruption of the benzene ring. ‘The “acetone bodies” (aceto-acetic acid, acetone and /3-hydroxybutyric acid) are then finally oxidized to carbon dioxide and water.

Neubauer, following the majority of other workers’ upon this subject, regards homogentisic acid as a normal product of the cata- bolism of tyrosine and of phenylalanine. Alcaptonuria is regarded as a condition in which there is simply a failure to deal with a normal product of intermediary metabolism, namely homogentisic acid.

From the results of experiments upon the fate of the deriva- tives of phenylalanine and of tyrosine in the normal and alcap- tonuric organism we are of the opinion that Neubauer’s repre- sentation of the normal catabolism of phenylalanine and of tyrosine will require considerable modification. The reasons for this opin- ion are as follows:

(1) No evidence has been put forward that phenylalanine undergoes hydroxylation in the aromatic nucleus under normal conditions. Injections of phenylalanine, so large that much appears unchanged in t’he urine, fail to evoke any excretion of phenolic substances including homogentisic acid.2

(2) It is improbable that tither a substance of quinonoid structure such as is represented by Neubauer as a precursor of homogentisic acid or homogentisic acid itself is formed in the course of the normal catabolism of phenylalanine and tyrosine.

‘For literature references see This Journal, viii, p. 11, 1910. Wf. Dakin: This Journal, vi, p. 235, 1909.

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142 Catabolism of Phenylalanine

The evidence for this is based upon the fact that para-methylpheny- Ealanine and para-methoxy-phenylalanine’ (tyrosine-methyl-ether) both substances which by reason of their constitution cannot be con- verted into para-quinonoid derivatives, undergo practically complete oxidation in the normal organism in precisely the same fashion as phenylalanine and tyrosine do.

(3) Para-methylphenylalanine and para-methoxy-phenylalanine, together with the corresponding ketonic acids, para-methyl-phenyl- pyruvic acid and para-methoxyphenylpyruvic acid, all of them sub- stances incapable of yielding para-quinonoid derivatives. yield aceto-acetic acid and acetohe when perfused through the surtiwing liver of the dog.

COOH COOH COOH COOH

I CHNH2

I

CHa OCH3 C& OCH, Para-methyl- Para-methoxy- Para-methyl- Para-methoxy- phenylalanine phenylalanine phenylpyruvic phenylpyruvic

Acid Acid

It is therefore clear that the series of reactions resulting in the pro- duction of aceto-acetic acid from tyrosine does not necessarily depend upon the prior formation of either a quinonoid intermediary substance or of homogentisic acid.

(4) The fact that substitution of the hydrogen atom in the para-position in phenylalanine by a methyl or methoxyl group does not interfere with its undergoing catabolism in the normal organism along similar lines to those of phenylalanine itself, is additional evidence supporting the view that phenylalanine is not necessarily converted into tyrosine in the course of its break- down in the animal body.

(5) The most convincing evidence that the type of change which

‘The experiments with para-methoxy-phenylalanine have already been reported. This Journal, viii, p. 11, 1910.

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A. J. Wakeman and H. D. Dakin I43

in the alcaptonuric results in the conversion of tyrosine into homogentisic acid is not the only route for the aromatic amino- acids to follow is found in the fact that if the previously mentioned synthetic amino-acids, para-methylphenylalanine and para-meth- oxy-phenylalanine are fed to an alcaptonuric, they are, within reasonable limits, completely oxidized. It therefore follows that even the alcaptonuric is provided with a mechanism for the oxi- dation of the aromatic nucleus of amino-acids, provided that their conversion into homogentisic acid is prevented by suitable sub- stitution in the para-position. (Cf. the following paper.)

Assuming that the above raised objections to Neubauer’s for- mulation of the normal course of catabolism of phenylalanine and tyrosine are sustained, the next step is obviously to try and sub- stitute some other scheme which is more in harmony wit’h the facts. The question at present practically assumes the form of trying to picture the conversion of tyrosine into aceto-acetic acid (since this change has been demonstrated by Embden and others), without the intermediary formation of homogentisic acid. In considering the formula for tyrosine the possibility presents itself that the carbon atoms adjacent to the hydroxyl group in the para-position might in the course of some molecular rearrangement be converted into fl-hydroxybutyric acid, which would in turn be oxidized to aceto-acetic acid and acetone.

COOH I

CHNHn

. . . + CHs.CHOH.CHz.COOH

This view is, however, untenable, in view of the formation of ace- to-acetic acid and acetone from amino-acids in which no hydroxyl group in the para-position can be introduced, owing to that position being already substituted. We are thus led to consider the portion of the tyrosine molecular to which the side-chain is attached. It is certain that the carboxyl group present in the tyrosine or phenylalanine molecule is not identical with that which

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I44 Catabolism of Phenylalanine

appears in the aceto-acetic acid molecule. Neubauer’s results indicate most clearly the intermediary formation of cr-ketonic acids followed by their oxidation with formation of an acid with one fewer carbon atom. When this is taken into account, along with the fact that aceto-acetic acid and acetone formation from phenylalanine derivatives is not inhibited by substitution in the para-position and furthermore the fact that benzene itself may be oxidized in the animal organism to muconic acid,l it appears practically certain that phenylalanine or tyrosine yields aceto- acetic acid in such a way that the carbon atom of the carbcxyl group and that in the cr-position in aceto-acetic acid are derived fr0.m the a! and p carbon atoms of the phenylalanine side chain while the re- maining p and y carbon atoms of the aceto-acetic acid molecule are derived from two adjacent carbon atoms in the benzene nucleus. The fact that phenylacetic acid does not yield aceto-acetic acid when perfused through a surviving liver may possibly be taken as indi- cating that disruption of the aromatic ring occurs prior to the oxidation of the ar-ketonic acid in the ar-position. At present we have not sufficient evidence to completely fill in the whole of the intermediate steps in the series of reactions but the scheme on the following page indicates the particular carbon atoms which it is believed are concerned in the formation of aceto-acetic acid.

Thisview of the formation of aceto-acetic acid furnishes an explan- aLion of the very different behavior of phenylalanine and of phenl- serine, C&f~.CHOH.CHNH~.COOH, in the animal body.2 The latter has been shown by one of us to yield hippuric acid when administered to cats and this failure to undergo complete oxida- tion in the body may fairly be ascribed to the impossiblilty of the formation of aceto-acet.ic acid from this substance owing to the presence of the hydroxyl group in the position adjacent to the benzene nucleus. The behavior of phenylserine in the body may be taken as additional evidence of the correctness of the sug- gestion put forward as to the mode of catabolism of phenylalanine and of tyrosine.

The fact that phenylamino-acetic acid and phenyl-a-amino- butyric acid in contrast to phenyl-a-aminopropionic acid (phengl-

‘Jaffe: Zeitschr. f. physiol. Chem., lxii, p. 58. 2This Journal, vi, p. 238, 1909.

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A. J. Wakeman and H. D. Dakin I45

alanine) do not undergo complete oxidation in the animal body, the benzene nucleus in both cases remaining intact is readily explained on the basis of t,he foregoing hypothesis as to the mode of catabolism of phenylalanine and tyrosine. Neither phenyl- amino-acetic acid nor phenyl-ac-aminobutyric acid can for struc- tural reasons yield aceto-acetic acid in the same way as phenyl- alanine and tyrosine do, if the suggested mechanism of the for- mation of the latter substance is correct.

COOH COOH COOH

I I I CHNHz co CO

I CHz CHz CHS -+

I

A /= g c=

;i y -+ “f : 7” -+ g

\c2H ““\,//“” )

H H (Phenylal- Phenylpyruvic Y

anine) Acid. CH

I c=

I H

(Phenylpyruvic Acid written in

open chain form.)

COz+HsO

COOH COOH

I I C& -+ CH2

I C= co -+ COz* + H,O I

;“- CHs (Aceto-

CH acetic

I Acid)

CH

II CH

I c= I

H

To sum up, it appears probable from a consideration of all t,he available evidence that of the nine carbon atoms in phenylalanine and tyrosine that present in the form of a carboxyl group is liber- ated in the form of carbon dioxide. Of the eight remaining carbon atoms, the four indicated in the accompanying diagram by heavier type form the carbon chain of an aceto-acetic acid molecule. It is possible that the remaining four carbon atoms,

*We have reason to believe that formic and probably acetic acid are inter- mediate products in the catabolism of aceto-acetic acid. The experiments bearing on this question will be published shortly.

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146 Catabolism of Phenylalanine

those occupying positions 2, 3, 4 and 5 in the aromat,ic nucleus, may undergo rearrangement with formation of a second molecule of aceto-acetic acid. At present there is no direct evidence bear- ing upon this latter point.

EXPERIMENTAL.

Liver PeTfusion Experiments. For the following experiments we made use of a very simple arrangement for the perfusion of the livers of dogs through the portal vein.

The animal was anesthetised with ether, morphine being avoided as apparently it had an injurious effect upon aceto-acetic acid formation by the liver. Artificial respiration was employed. The portal vein was then exposed for a short distance close to its entrance to the liver and two loose ligatures placed around the vein. Care was taken to ligature any tributaries entering the vein in close proximity to the liver. A third loose ligature was placed around the vena cava above the entrance of the renal veins. The animal was then completely bled from a cannula previously inserted in the carotid or femoral artery. The blood was whipped and added to the perfusion fluid which had been previously pre- pared. As soon as the bleeding was practically complete the liga- ture around the vena cava was tied, also the ligature around the portal vein, farthest removed from the liver. A large, long glass cannula was then inserted in the portal vein and connected by a rubber tube to a syphon placed in the flask containing the warmed perfusion fluid. Another cannula was tied in the vena cava between the heart and diaphragm. A rubber tube attached to the cannula carried away the perfusion fluid from the liver and delivered it into a large flask which was kept at a temperature of about 38 degrees. Under favorable conditions perfusion was commenced in less than five minutes after the death of the animal. The first portions of blood coming from the liver were separately collected, whipped and returned to the main supply. Oxygen was bubbled through the liquids in both the delivering and receiving flasks so that the blood was well oxygenated. The fluid after travers- ing the liver was returned to the delivery flask. The rate of per-

‘This Journal, vi, p. 238, 1909.

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A. J. Wakeman and H. D. Dakin I47

fusion ordinarily varies from a minimum of 70 cc. per minute to about 200 cc. per minute. The pressure varied from about 2.5-5 cm. of mercury and was kept as low as possible. There appear to be some advantages in perfusing the liver in situ as described, rather than to excise the organ. The animal’s body was kept warm throughout the experiment. The perfusion ordi- narily lasted one hour.

The perfusion fluid was composed of a mixture of defibrinated bullock’s blood together wit.h t,he dog’s own blood, and a solution of the substance under investigation in 50-100 cc. of saline. The amino-acids were relatively sparingly soluble in cold salt solution so t’hat the following method was employed for preparing them in solution. Two grams of the amino-acid, e. g., para-methylphenyl- alanine or para-methoxyphenylalanine, were converted into the readily soluble hydrochlorides by warming with 50 cc. of 9” hydrochloric acid. An amount of normal caustic soda solution (10 cc.) sufficient to exact,ly neutralize the hydrochloric acid was very rapidly added, followed immediately by 500 cc. of blood. In this way, the amino-acid has practically no opportunity of crystallizing out and is readily obtained in neutral solution dis- solved in blood slightly diluted with salt solution. The ketonic acids investigated, para-methyl-phenylpyruvic acid and para- methoxy-phenylpyruvic acid were converted into their readily soluble neutral ammonium or sodium salts.

The aceto-acetic acid and acetone estimations were in most cases made by taking an aliquot part of the blood after perfusion, diluting freely with water acidified with phosphoric acid and dis- tilling in capacious flasks after making liberal additions of paraffin wax to control the foaming. In one or two cases the blood was treated with Schmidt’s mercuric chloride reagent before distilla- tion but the results were similar in either case. The acetone present in the distillates was estimated by means of iodine solu- Con in the usual way.

The principal results of the experiments are recorded in the following table.

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14s

NO.

--

I II

III

IV V

VI VII

VIII IX u

Catabolism of Phenylalanine

Kilo

30 30 23 28 31 14 32 15 15

Blank Blank Blank

Phenylalanine Para-methyl-phenylalanine Para-methyl-phenylalanine Para-methyl-phenylalaninc Para-methoxy-phenylalanine Para-methoxy-phenylalanine Para-methyl-phenylpyruvic

Acid Para-methyl-phenylpyruvic

Acid Para-methoxy-phenylpyruvic

Acid

grams

0 0 0

2.0 2.0 2.0 2.0 2.0 1.5 2.0

2.0

2.0

/PI

I- I FORMED

minutes 1 mizzigram!

;; 1 f.2

60 ~ 32.5 60 166 60 1 84.2 85 ~ 78.4 60 153 80 186 60 98 60 1 62.5

60 1 116

75 119

The results of the experiments require little or no comment. It will be seen that definite positive indications of the formation of aceto-acetic acid, were obtained when each of the four para- substituted substances: p-methyl-phenylalanine, p-methoxy- phenylalanine, p-methyl-phenylpyruvic acid, p-methoxy-phenyl- pyruvic acid, was added to the blood used for perfusing the liver. An experiment confirmatory of Embden’s results with phenylala- nine is also included in the table.

The details of the preparation of the substances used for per- fusion are given in the following pages.

Para-methyl-phenylalanine (Para-tolylalanine). This amino-acid was prepared from para-methylbenzaldehyde by a method sim- ilar to Erlenmeyer and Halsey’s synthesis of phenylalanine. The details of the preparation are given in the following paper. The amino-acid crystallizes from water in prisms melting at 277-279.”

Para-methoxy-phenylalanine (Tyrosine methyl ether). The syn- thesis of this amino-acid has already been described.l The last step in the preparation involving the hydrolysis of the benzoyl derivative of the amino-acid is apt to result in t,he formation of a

IThis Journd, viii, p. 17, 1910.

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A. J. Wakeman and H. D. Dakin I49

small amount of tyrosine as the result of the action of the hydro- chloric acid upon the methoxyl group. It is important therefore not to use too concentrated hydrochloric acid for the hydrolysis. A specimen of para-methylphenylalanine which gave a distinct Millon reaction due to contamination with a trace of tyrosine was purified as follows. The amino-acid was dissolved in eight parts cold dilute nitric acid, sp. gr., 1.15 and allowed to stand over night. A small separation of nitro-tyrosine occurred while the methoxy- phenylalanine was not nitrated under these conditions. The fil- trate was neutralized with ammonia, concentrated in the water- bath and the amino-acid allowed to crystallize out. It was then washed with a little cold water and recrystallized from boiling water. The resulting methoxy-phenylalanine was perfectly free from tyrosine and gave no reaction with Millon’s reagent.

Para-methylphenylpyruvic acid. This acid has apparently not been previously described. It was prepared by Plochl’s method by heating the product of the condensation of para-methylben- zaldehyde and hippuric acid with caustic soda solution. The con- densation product was obtained by heating on the water-bath for half an hour a mixture of para-methylbenzaldehyde (1 mol.), powdered hippuric acid (1 mol.), powdered fused sodium acetate (1 mol.) and acetic anhydride (2 mols.) The yellow “azlactone” readily separates out and is purified by washing wit,h water and recrystallizing from alcohol. The substance melts at 141-142’. (Cf. following paper.) On boiling the “azlactone” (15 gms.) with fifteen times its weight of 40 per cent caustic soda solution for an hour, ammonia is freely evolved. On cooling and acidifying with a mixture of equal parts of crushed ice and concentrated hydro- chloric acid a precipitate of impure benzoic acid is at once obtained. This precipitate is filtered off after a few minutes and the filtrate placed in a cool place. The crude ketonic acid slowly separates out of solution on long standing in a cool place and is purified by recrys- tallization from water containing a little alcohol. The substance crystallizes in prismatic needles, m. p., 178-180,0 is readily soluble in alcohol and in ether. Its alcoholic solution gives a blue-green coloration on addition of ferric chloride.

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150 Catabolism of Phenylalanine

ANALYSIS : 0.1197 gm. gave 0.2981 gm. COz and 0.0575 gm. HzO.

Found: Cal&$; for

Carbon......................................... 67.9 67.4 Hydrogen...................................... 5.4 5.6

Para-methoxy-phenylpyruvic-acid. This acid has apparently not been previously described. It was prepared in precisely the same manner as the preceding ketonic acid by heat,ing the conden- sation product of anisaldehyde and hippuric acid with strong caustic soda. This “azlactone” has already been described.1 The ketonic acid slowly separates out from solution in the form of opaque needles. The acid is purified by repeated recrystalliza- tion from boiling water and melts at 199-192 degrees. It is very readily soluble in alcohol and-ether, moderately soluble in boiling water, sparingly soluble in cold water. The alcoholic solution of the substance gives a deep blue coloration on addition of ferric chloride.

ANALYSIS: 0.1275 gm. gave 0.02910 gm. COz and 0.0574 gm. H20 Calculated for

Found: CwH1o0~: Carbon......................................... 62.2 61.9 Hydrogen...................................... 5.0 5.1

‘This Journal, viii, p. 19, 1910.

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A. J. Wakeman and H. D. DakinTHEIR DERIVATIVES

PHENYLALANINE, TYROSINE AND OF THE CATABOLISM OF

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