Homogentisic acid tyrosine oxidation

C. Excreted in the urine in the rare hereditary disease alkaptonuria. Homogentisic acid is easily oxidized in the air to dark-coloured polymeric products, so that urine from patients with alkaptonuria turns gradually black. It is formed from tyrosine and is an intermediate in tyrosine breakdown in the body. Alkaptonuria is due to the absence of the liver enzyme which cleaves the aromatic ring. 

The fundamental role of ascorbic acid in metabolic processes is not well understood. There is some evidence that it may be involved in metabolic hydroxylation reactions of tyrosine, proline, and some steroid hormones, and in the cleavage-oxidation of homogentisic acid. Its function in these metabolic processes appears to be related to the ability of vitamin C to act as a reducing agent. 

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... 

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. 

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 homogentisic acid were normal intermediates in tyrosine metabolism, and although homogentisic acid was known to be readily metabolized by normal liver (e.g., 208, 695, 976) Felix and co-workers (262) considered p-hydroxyphenylpy-ruvic acid and homogentisic acid not to be intermediates in the breakdown of tyrosine by the liver system. 

The amino acids phenylalanine and its hydroxylated derivative, tyrosine, are both catabolised in the livers of animals to fumaric acid and acetoacetic acid via homogentisic acid. This is formed by the oxidation of 4-hydroxyphenylpyruvate, catalysed by the copper containing enzyme 4-hydroxyphenylpyruvate dioxygenase, which requires vitamin C for its activity. The complete sequence is shown in Figure 5.13. The dioxygenase is so called because both the atoms of the... 

Tyrosine Catabolism - The catabolism of tyrosine to fumarate and acetoacetate is depicted in Figure 21.21. A hereditary deficiency of the enzyme homogentisic acid dioxygenase causes a disease called alkaptonuria where homogentisic acid accumulates and is excreted in large amounts in the urine. It oxidizes on standing, causing the urine to become dark. 

La Du, 8.N. Zannoni, V.G. The Tyrosine Oxidation System of Liver II Oxidation of p-Hydroxylphenylpyruvic Acid to Homogentisic Acid (1955) 217 777-787... 

Phenylalanine is ultimately catabolized to acetoacetate and fumarate, so it is both ketogenic and glucogenic. Initially, phenylalanine is hydroxylated to tyrosine, then transaminated to j5-hydroxyphenylpyruvate. This is farther oxidized to homogentisic acid and then to fumarate and acetoacetate. What happens to a patient if the enzyme that is required for the cleavage of the aromatic ring is absent ... 

Although plants and bacteria do not have the ability to do so, most animals can convert phenylalanine to tyrosine (Haslam, 1974). C6-C2 compounds are probably formed by a-oxidation of ketoacids such as phenylp)nuvic (10) and p-hydroxypyruvic acid (11). One well-known example, phen-ylacetic acid (8), is common in plants (Gross, 1981). Mammals and some microorganisms convert phenylalanine and t5rrosine to another C6-C2 compound, homogentisic acid (9) (Fig. 8.3). 

In alkaptonuria, homogentisic acid (28) is excreted in excessive amounts indicating that in this metabolic disease the opening of the aromatic ring and complete oxidation to CO2 and H2O is blocked. In albinism, there is a deficiency of the enzyme systems responsible for the formation of melanin from tyrosine (24) (Figure 3.7). 

Studies on alcaptonurics have been particularly informative, since these patients excrete homogentisic acid in their urine and are apparently unable to oxidize this acid further. Assuming that homogentisic acid is a normal intermediate of tyrosine oxidation, various compounds have been fed to alcaptonurics in order to discover whether they were an intermediate in the pathway preceding the formation of homogentisic acid. From the result of such studies a scheme of the catabolism of phenylalanine and tyrosine has been developed which has been extended and supplemented by other types of experiments, particularly in recent years by experiments wdth isotopic tracers and liver slices and enzyme preparations. 

Recently extracts of acetone powder and homogenates of liver have been obtained which specifically oxidize tyrosine, p-hydroxyphenyl-pyruvic acid, and homogentisic acid to acetoacetic acid. The reaction is aerobic and requires the uptake of 4 atoms of oxygen per molecule for the oxidation of either L-tyrosine or p-hydroxyphenylpyruvic acid to yield 1 molecule of CO2 and 1 of acetoacetic acid oxidation of homogentisic acid to acetoacetate requires the uptake of 2 atoms of oxygen. 

Pyruvate inhibits the oxidation of tyrosine and p-hydroxyphenyl-pyruvate, but not of homogentisic acid. This inhibition can be reversed by ascorbic acid or hydroquinone and its site of action, then, appears to be in relation to 2,5-dihydroxyphenyl formation from p-hydroxyphenyl-pyruvate. 

The oxidative breakdown of the amino acid tyrosine contains a stage in which homogentisic acid is converted to maley-lacetoacetic acid, a reaction catalysed by homogentisic acid oxidase. A deficiency of this enzyme results in the accumulation... 

An organic acid found in the urine of patients with alkaptonuria, a hereditary condition in which there is a deficiency of homo-gentisic acid oxidase, one of the enzymes of tyrosine metabolism. As a result, homogentisic acid accumulates in the blood, tissues and urine. Urines containing homogentisic acid turn dark on standing due to oxidation and polymerization of the compound to the black pigment alkapton. 

The oxidative metabolism of L-phenylalanine and L-tyrosine is of intrinsic interest from the medical point of view since a number of diseases resulting from inborn errors of metabolism are associated with this pathway. Some indication of the intermediates in the pathways of oxidative metabolism of L-phenylalanine and l-tyrosine indeed came first from a study of one such disease, alcaptonuria, in which the patient excretes homogentisic acid. Garrod recognised that these errors of metabolism were due to a congenital inability of the organism to carry out a particular enzyme transformation in the normal sequence of events it is a natural mutant. 

A rare inborn (recessive) abnormality of metabolism in man marked by the inability to complete the degradation of tyrosine and phenylalanine their metabolism ceases at homogentisic acid, which is excreted in the urine. The homogentisic acid oxidizes to black melanoid pigment hence, the urine of alcaptonurics slowly turns black. The defect appears to be harmless. 

Five enzyme steps have been demonstrated to be required in the conversion of tyrosine to fumaric and acetoacetic acid. These consist of a transamination to p-hydroxyphenylpyruvate, a simultaneous oxidation, migration of the side chain and decarboxylation to form homogentisic acid, oxidation of the latter to maleylacetoacetic acid, isomerization of this compound to fumarylacetoacetic acid, and hydrolysis of this acid to fumaric acid and acetoacetic acid. 

An interesting aspect of the oxidation of tyrosine has been the role of ascorbic acid 212, 214-219). Incomplete tyrosine metabolism with the excretion of homogentisic acid and other incomplete oxidation products... 

The next enzymic step in the dissimilation of tyrosine, oxidation of homogentisic acid, was clearly established by the work of Ravdin and Crandall 2H0). These investigators isolated two enzyme fractions from a rat liver homogenate, one of which catalyzed the oxidation of homogentisic acid to an open chain diketone-dicarboxylic acid. In their hands the product isolated was 4-fumarylacetoacetic acid. Subsequent work has shown that the initial product is 4-maleylacetoacetic acid and that an isomerase is present which converts this to the fumarylacetoacetate 221). The second enzyme of Ravdin and Crandall 220), fumarylacetoacetic acid hydrolase, hydrolytically cleaves this compound to fumarate and aceto-acetate. 

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