P-Hydroxyphenylpyruvate oxidase

Figure 20.22 Catabolism of phenylalanine and tyrosine. A indicates the lesion in classic phenylketonuria B indicates a tyrosinemia caused by tyrosine transaminase deficiency C indicates a tyrosinemia caused by p-hydroxyphenylpyruvate oxidase deficiency and the lesion in neonatal tyrosinemia D indicates alcaptonuria.

Additional errors of phenylalanine and tyrosine metabolism include tyrosinosis, or hereditary tyrosinemia, neonatal tyrosinemia, and alcaptonuria. In the first case, there is a probable defect in p-hydroxyphenylpyruvate oxidase. In neonatal tyrosinemia, the problem is transient and may be solved by the administration of ascorbic acid. Ascorbic acid is apparently a cofactor for p-hydroxy-phenylpyruvate oxidase. Alcaptonuria is a benign disorder in which homogen-tisic acid oxidase is inoperative and homogentisic acid is excreted in the urine. Air oxidizes the homogentisic acid to a pigment, giving urine a black color. This pigment also accumulates in the patient s tissues. 

Tyrosine Oxidation (p-Hydroxyphenylpyruvate Oxidase) (Eq. 17) The interruption of tyrosine degradation at the stage of p-hydroxy-phenylpyruvate (pHPP) in scurvy has long been considered Ae clearest evidence of a biochemical role for ascorbic acid. The intmmediate accumulates and is excreted when doses of tyrosine are given to scorbutic man, other primates, and guinea pigs, and to premature infants (K8). 

Fig. 5. Changes of liver enzyme activities after tyrosine dosage of guinea pigs tyrosine-a-ketoglutarate transaminase (- -o- -), p-hydroxyphenylpyruvate oxidase (— x—). Each point is the average of homogenates from two animals. From Knox and Goswami (Kll).

Purification of p-hydroxyphenylpyruvic oxidase led to the separation of two proteins, neither of which is active alone. One of these was identified as catalase. Catalase can be replaced as a component of the oxidase system by a peroxidase. These findings implicate H2O2 in the oxidation, and this is supported by the elimination of a lag period in the reaction by the addition of small quantities of H2O2. In spite of the requirement for two proteins and the effect of H2O2, an intermediate-level oxidant, no... 

A single copper containing protein with a requirement for ascorbic acid—p-hydroxyphenylpyruvate oxidase—catalyses the simultaneous hydroxylation of the phenyl ring, migration of the aliphatic side chain to an adjacent position on the ring, and oxidative decarboxylation of the pyruvate fragment . The enzyme... 

The oxidation was also strongly inhibited by diethyldithiocarbamate and this can be partially reversed by small amounts of Cu". The apparent requirement for Cu", was supported by the observation that the livers of copper-deficient dogs had about half the p-hydroxyphenylpyruvate oxidase activity of littermate controls. However, this lowered activity due to copper deficiency could not be increased by addition of exogenous Cu++. 

The results of LaDu and Zannoni on dog liver enzyme were substantiated by Hager c< al. 212) on enzyme preparations from beef and pig liver The enzyme from these sources were purified by ethanol precipitation, (NH4)2S04 precipitation, and chloroform-ethanol treatment. A purification of about a hundredfold was achieved. These authors suggest that p-hydroxyphenylpyruvate oxidase is a copper enzyme with properties similar to tyrosinase. Additional support for this is the relative insensitivity of the enzyme to CO and CN and the very high affinity for O2. 

Phenylalanine hydroxylase is like p-hydroxyphenylpyruvate oxidase (see below) in its requirement for two enzyme components. It is particularly interesting that one of these components (Fraction II) can be replaced by acetone powder extracts of liver from rabbit, calf, dog, and pig, and by acetone powder extracts of kidney and heart from calf and hog, although these oi ans contain no phenylalanine hydroxylase activity. Fraction II appears to be wide distributed, and to have fimctions other than these connected with phenylalanine hydroxylation. Although two enzyme components are involved in the system, no evidence for an intermediate between phenylalanine and tyrosine has been found (557), nor has it been possible to separate the process into two steps. 

Systems which oxidize p-hydroxyphenylpyruvate have been found in hog liver (224,239), rabbit liver (766), rat liver (429,465,810) and dog liver (466,838). Soluble, mitochondria-free p-hydroxyphenylpyruvate oxidase from rat liver oxidizes 2,5-dihydroxyphenylpyruvic acid to acetoacetate at a rate comparable with that of the over-all oxidation of L-tyrosine (429), but will not oxidize 2,5-dihydroxyphenyl-alanine, gentisic acid, or the lactone of 2,5-dihydroxyphenylpyruvic acid (429). A purified rabbit liver oxidase transforms p-hydroxy-... 

Partial purifications of p-hydroxyphenylpyruvate oxidase have recently been reported (224,468,469), but results of these studies are... 

Ascorbic acid has been implicated in tyrosine metabolism (Knox, 1955). A relatively specific effect of ascorbic acid has been demonstrated in the maintenance of activity of p-hydroxyphenylpyruvic oxidase. In some cases ascorbic acid has been used to support homogentisic oxidase also, but here the effect seems to be less specific, and probably is only to maintain the iron in the reduced, ferrous state (Suda et al., 1951 a, b). 

The metabolism of phenylalanine will now be considered in some detail, as two inborn errors of metabolism are known that affect this pathway. Phenylalanine is first hydroxylated by phenylalanine hydroxylase to form another aromatic amino acid tyrosine (Fig. 8). The coenzyme for this reaction is the reductant tetrahydrobiopterin which is oxidized to dihydrobiopterin. Phenylalanine hydroxylase is classified as a monooxygenase as one of the atoms of 02 appears in the product and the other in HzO. The tyrosine is then trans-aminated to p-hydroxyphenylpyruvate, which is in turn converted into homogentisate by p-hydroxyphenylpyruvate hydroxylase. This hydroxylase is an example of a dioxygenase, as both atoms of 02 become incorporated into the product (Fig. 8). The homogentisate is then cleaved by homogentisate oxidase, another dioxygenase, before fumarate and acetoacetate are produced... 

Z2. Zannoni, V. G., and La Du, B. N., Tyrosyluria resulting from inhibition of p-hydroxyphenylpyruvic acid oxidase in vitamin C-deficient guinea pigs. /. Biol. Chem. 236, 2667-2671 (1960). 

A rare inborn error of metabolism in which there is excessive urinary excretion of tyrosine andp-hydroxyphenylpyruvicacid, an intermediate in the catabolism of tyrosine. The enzyme thought to be deficient is p-hydroxyphenylpyruvicacid oxidase, the enzyme which converts p-hydroxyphenylpyruvic acid to... 

In the metabolism of tyrosine, a deficiency of vitamin C will result in the build-up and excretion of the intermediary product, P-hydroxyphenylpyruvate, as a result of inactivating the enzyme P-hydroxyphenylpyruvic acid oxidase. When large amounts of tyrosine are being metabolized, vitamin C protects the enzyme P-hydroxyphenylpyruvic acid oxidase from inactivation (rather than activates as was formerly thought), and enhances the synthesis of norepinephrine, a neurotransmitter, from tyrosine. 

On the other hand, an enzyme system purified 100-fold from hog liver oxidizes p-hydroxyphenylpyruvate but not 2,5-dihydroxyphenylpyruvate (224). The corresponding purified dog liver oxidase will not oxidize 2,5-dihydroxyphenylpyruvate, nor will it attack o-hydroxyphenylpyruvate, 7 i-hydroxyphenylacetate, p-hydroxyphen-ylacetone, or the lactone of 2,5-dihydroxyphenylp3rruvic acid (467). Furthermore, one molecule of Os is consumed per molecule of p-hydroxyphenylpyruvate and per molecule of COs evolved. These ratios are 1 throughout the course of the oxidation no accumulation of intermediate can be detected by this means (469,470). 

---