Homogentisate oxidase

Answer A. Adults with alcaptonuria show a high prevalence of ochronotic arthritis due to deficiency of homogentisate oxidase. 

Homogentisate oxidase catalyzes an important reaction In tyrosine metabolism, which converts the substrate homogentisic acid to the product maleylacetoacetic acid. 

Inborn errors of metabolism are inherited metabolic disorders caused by the absence of an enzyme in a metabolic pathway. Alkaptonuria is caused by the lack of homogentisate oxidase and is harmless, whereas phenylketonuria, which is due to a lack of phenylalanine hydroxylase, can cause severe mental retardation. 

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... 

The major quantitative pathway of tyrosine catabolism produces acetoacetate and fumarate. If homogentisate oxidase is missing, the result is alcaptonuriadark urine. 

Alcaptonuria occurs when homogentisate, an intermediate in tyrosine metabolism, cannot be further oxidized because the next enzyme in the pathway, homogentisate oxidase, is defective. Homogentisate accumulates and auto-oxidizes, forming a dark pigment, which discolors the urine and stains the diapers of affected infants. Later in life, the chronic accumulation of this pigment in cartilage may cause arthritic joint pain. 

Homogentisate oxidase Homogentisic acid Alcaptonuria Black urine, arthritis... 

Alkaptonuria is an autosomal recessive, benign disorder with a normal life expectancy. It is caused by a deficiency of homogentisate oxidase (Fig. 47.2). Homogentisate accumulates, is excreted in the urine and is gradually oxidised to a black pigment when exposed to air. It is usually detected when the nappies (or diapers) show black staining. 

All known dioxygenases contain iron, either in heme groups or as Fe-S centers. Some also contain copper. Examples are 1Vyptophan-2,3-dioxygenase (see), homogentisate oxidase (see L-Phenylalanine), 3-hydroxy-anthranilate oxidase (see L-llyptophan). 

Homogentisic acid is oxidized by a ferrous ion-requiring enzyme, as shown by Suda and Takeda, and confirmed by several others. Homogentisic oxidase also requires a sulfhydryl compound. One molecule of oxygen is consumed in the reaction, presumably in a single step. The product has been identified as maleylacetoacetate by Knox and Edwards (V). ... 

The dioxygenases, which incorporate two atoms of oxygen into one molecule of the substrate, are enzymes which are frequently involved in the cleavage of bonds in an aromatic ring. Typical of these are homogentisate oxidase and L-tryptophan oxidase (L-tryptophan pyrrolase) and two bacterial oxygenases pyrocatechase and metapyrocatechase. ... 

Formation of maleylacetoacetate upon incubation with homogentisic oxidase is detected by the difference in its absorption spectrum from that of fumarylacetoacetate. The former at pH 3 has an absorption maximum at 230 mu, while the latter compounds has its maximiun at 350 m. At pH 1, fumarylacetoacetate exhibits maximum absorption at 310 m, while maleylacetoacetate has almost no absorption. The difference in spectra is accoimted for by the fact that fumarylacetoacetate exists mainly in the enol form in acid solutions, while maleylacetoacetate is laigely in the keto form. Enol formation is enhanced by borate which leads to the formation of an enol fumarylacetoacetate-borate complex. 

The properties of homogentisate oxidase have been studied by several investigators. Suda and Takeda found that, in the final stages of enzyme preparation (from rabbit liver), a sharp drop in activity occurs upon salting-out (715). This drop can be reversed by ferrous iron, a requirement which has been repeatedly confirmed (169,436,647,720). The activation effect of iron is potentiated by ascorbic acid, but ascorbate can be replaced by other enediols (646). When purified preparations are aged or incubated at pH 5.5-6.0, activity is lost. It cannot be restored by ascorbate, but can be restored by ferrous iron although ferrous ascorbate is more effective than ferrous iron alone. Other metals fail to reactivate the enzyme (Mg++, Mn++, Zn++, Co++, or A1+++), and ferrous iron has no effect upon fresh preparations (169). Hie inhibition that occurs when the pH is raised above the optimum of the enzyme (around 7.0) is readily reversible by restoring the pH to 7.0, but the inhibition that occurs when the pH is lowered to 6.0 is not reversible in this... 

Fig. 3. The pH-activiiy curve of homogentisate oxidase alone, and homogenti-sate oxidase supplemented with ferrous ion and ascorbate (171). Solid circles, ensyme and substrate alone. Open circles, enzyme and substrate supplemented with 10 M Fe" and 6 X 10 Af ascorbate.

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