Biochemistry of Amino Acids - A. Meister 1961

Intermediary Metabolism of Amino Acids
Phenylalanine and Tyrosine
Phenylalanine and Tyrosine Metabolism in Animals

It has long been known that The breakdown of Phenylalanine and Tyrosine in animal organisms yields acetoacetic acid. The elucidation of this pathway began with studies on certain inborn errors of human METABOLISM (see Chapter V). The identification of the intermediate reactions in this process has made significant progress in recent years through studies utilizing labeled metabolites and various enzyme preparations. Experimental evidence regarding The excretion of homogentisic acid in patients with alkaptonuria, the elevated excretion of homogentisic acid following the ingestion of phenylalanine and tyrosine [930], and The formation of acetoacetic acid from homogentisic acid in the perfused Liver [931, 932] provided a basis for the hypothesis that homogentisic acid acts as an intermediate in the metabolism of aromatic Amino Acids. It was established that in normal animals, homogentisic acid, like phenylalanine and tyrosine, undergoes oxidation to yield acetoacetic acid among other products. When animals are fed large amounts of phenylalanine and tyrosine, an excretion of homogentisic acid is observed [933–938].

The conversion of phenylalanine into tyrosine was established as early as 1909 by Neubauer [1930]; it is now known that this reaction plays a quantitatively major role in animals. In 1913, Embden and Baldes [931] observed the formation of tyrosine from phenylalanine in liver perfusion experiments. In a subject suffering from tyrosinosis, the administration of phenylalanine resulted in an increased urinary excretion of tyrosine [939]. The reduction in the dietary requirement for phenylalanine upon the inclusion of tyrosine in the diet has already been mentioned (p. 121). Furthermore, it is known that premature infants suffering from C-hypovitaminosis excrete tyrosine following the administration of phenylalanine [940, 941]. All these data point to the conversion of phenylalanine into tyrosine. This process was definitively proven in experiments on rats by feeding them deuterium-labeled phenylalanine; tyrosine with the corresponding label was isolated from the animal Tissues [942]. In patients suffering from Phenylpyruvic Oligophrenia, this reaction proceeds to a very limited extent (p. 475).

Udenfriend and Cooper [943] achieved the conversion of phenylalanine to tyrosine in vitro using an enzyme system obtained from the liver (phenylalanine hydroxylase). The Mechanism of this reaction is complex; apparently, it requires two protein fractions, diphosphopyridine nucleotide, an aldehyde, oxygen, and Fe⁺⁺ ions [923, 1134]. The results of experiments using labeled tyrosine demonstrated that the reverse conversion of tyrosine to phenylalanine does not occur in animal organisms [944]. The process of converting phenylalanine to tyrosine has been observed in both Muscle and liver tissue [224]. Some microorganisms are capable of catalyzing the formation of tyrosine from phenylalanine [923], but most Bacteria apparently lack the corresponding enzyme system.

The conversion of phenylalanine into acetoacetic acid has been studied in experiments utilizing isotopic carbon [946–951]. These elegant and conclusive studies demonstrated that: a) the a-carbon atom of phenylalanine becomes the carbon of the carboxyl group of acetoacetic acid, b) the C-2 atom of the benzene ring serves as the precursor of the carbonyl carbon atom of acetoacetic acid, and c) the C-1 or C-3 atoms of the ring serve as precursors of the carbon atom of the methyl group of acetoacetic acid. Other studies [947–950] found that the β-carbon atom of tyrosine becomes the a-carbon atom of acetoacetic acid. These findings indicate a rearrangement of the side chain during The oxidation of phenylalanine and tyrosine. It was established that the breakdown of tyrosine and phenylalanine yields two four-carbon fragments; one of these is represented by Ketone Bodies, and the other by malic acid or a closely related compound [951]. The data outlined above can be represented by the following scheme:

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The reaction sequence described above was confirmed by results from experiments with purified Enzymes, which revealed additional intermediates. The First stage of tyrosine conversion consists in the formation of p-hydroxyphenylpyruvic acid via a Transamination reaction. Interestingly, earlier studies using liver mince did not observe Ammonia Formation during the oxidation of tyrosine. Transamination between a-ketoglutaric acid and tyrosine (Table 21) is required for subsequent oxidation reactions [952–954]. The possibility of tyrosine oxidation proceeding via 2,5-dihydroxyphenylalanine is evidently ruled out, as it has been established that this compound is not oxidized by the enzyme system responsible for tyrosine oxidation [953].

However, in the bodies of patients with alkaptonuria, 2,5-dihydroxyphenylalanine can be converted into homogentisic acid [955]. In vitro experiments show that p-hydroxyphenylpyruvic acid is rapidly oxidized to yield the same products as those formed during the oxidation of tyrosine itself [953].

Data have been published indicating that ascorbic acid [953, 956–962] and catalase [1135] play a definite role in the oxidation of p-hydroxyphenylpyruvic acid to homogentisic acid. The mechanism of ascorbic acid action remains unclear; it can be replaced in experiments by certain Other Compounds, such as isoascorbic acid. There is disagreement regarding whether 2,5-dihydroxyphenylpyruvic acid is an intermediate in the oxidation of p-hydroxyphenylpyruvic acid. It has been reported that 2,4-dihydroxyphenylpyruvic acid accumulates during the oxidation of p-hydroxyphenylpyruvic acid [959]. Later studies found that 2,5-dihydroxyphenylpyruvic acid is not oxidized at a noticeable rate by the enzyme system that converts p-hydroxyphenylpyruvic acid into homogentisic acid [961].

Under conditions of ascorbic acid deficiency, animals given oral doses of large amounts of phenylalanine or tyrosine excrete p-hydroxyphenylpyruvic acid in their urine; The amount of excreted acid decreases upon the administration of ascorbic acid to such animals [934–938]. Interestingly, pteroylglutamic acid prevents the urinary excretion of p-hydroxyphenylpyruvic acid induced in guinea pigs by feeding them tyrosine, yet it does not protect the animals against scurvy [963]. The oxidation of tyrosine in liver preparations obtained from Folic acid-deficient rats could be reactivated by the in vitro addition of folic acid [964, 965].

Experiments utilizing isotopes have demonstrated that the conversion of p-hydroxyphenylpyruvic acid to homogentisic acid is accompanied by a side-chain rearrangement. This reaction is evidently analogous to the oxidation of p-cresol to methylhydroquinone:

It is possible that the corresponding biological reaction proceeds in a similar manner [966, 967]; the mechanism of this transformation and the function of ascorbic acid within it [968, 969] require further study.

The oxidation of homogentisic acid has been studied in various in vitro systems [970–976]. The enzyme involved in this process has been found in The Liver and Kidneys, as well as in a strain of Pseudomonas; its activity requires the presence of Fe⁺⁺ ions, whereas Fe⁺⁺⁺ ions are inactive. Ascorbic acid and Glutathione activate the system—possibly by protecting Fe⁺⁺ and sulfhydryl groups from oxidation—while a,a'-dipyridyl inhibits homogentisic acid oxidation, most likely by chelating the iron. The action of this highly unusual enzyme system consumes one mole of oxygen and yields maleylacetoacetic acid. This product, whose formation was initially suspected on The basis of ultraviolet absorption spectra [977], is converted into fumarylacetoacetic acid by an enzyme that requires glutathione as a cofactor [1136, 1137]. Interestingly, only fumarylacetoacetic acid, but not maleylacetoacetic acid, is hydrolyzed by a soluble liver enzyme [970] previously identified by its ability to hydrolyze compounds such as a,y-diketo acids and β,δ-diketohexanoic acid [978–980]. The oxidation of homogentisic acid and the subsequent phases of its metabolism can be represented as follows:

Three human DISEASES ASSOCIATED WITH inborn errors of phenylalanine oxidation have been described; these are discussed in Chapter V.

The reactions detailed above represent the primary pathway of phenylalanine metabolism in mammalian organisms, although other metabolic routes for phenylalanine are also known. It has been established, for example, that normal human urine contains phenylacetylglutamine [981, 982] and that the excretion of this compound is significantly elevated following the administration of phenylacetic acid [983–989]. Upon the administration of 20 g of phenylacetic acid, almost the entire dose is excreted as phenylacetylglutamine. In all animal species investigated (dog, rat, rabbit, monkey, horse, sheep, and cat), phenylacetic acid loading led to the urinary excretion of phenylacetylglycine. Only in humans and, presumably, chimpanzees is phenylacetylglutamine formed. The appearance of this compound in urine suggests the occurrence of the following reactions:

Another metabolic pathway for phenylalanine involves its conversion into hippuric acid. Although hippuric acid excreted by humans and animals is derived principally from dietary benzoic acid, its presence in the urine of fasting humans [982], as well as data from studies utilizing labeled benzoic acid [990] and labeled phenylalanine [991], indicate that a portion of the urinary hippuric acid is of endogenous origin (e.g., derived from phenylalanine).