The enzymatic conversion of homogentisic acid to 4-fumarylacetoacetic acid.

Although the probability that homogentisic acid is an intermediate in the conversion of phenylalanine and tyrosine to acetoacetate is indicated by many experiments with humans (l-5), animals (6-9), and tissue preparations (lO-13), no direct evidence has been reported concerning the mechanism of its oxidation. However, the formation of /?-ketoadipic acid from phenol in the presence of cholera Vibrio, reported by Kilby (14), and the demonstration by Stanier et al. (15) that this acid is formed when either catechol or protocatechuic acid is oxidized by Pseudomonas &orescens suggest the possible conversion of homogentisic acid to a long chain /3-keto acid by oxidative cleavage of the aromatic ring at one point. The demonstration by Lerner (16) that phenylalanine is converted to malic and acetoacetic acids in the mammalian liver led him to postulate the conversion of homogentisic acid to an g-carbon dicarboxylic acid. Our preliminary experiments on the oxidation of homogentisic acid by rat liver homogenates showed the entire activity to be located in the clear supernatant fluid obtained by centrifuging the homogenate at approximately 140,000 X g for 45 minutes at 0”. Oxygen consumption, homogentisate disappearance, and acetoacetate formation were essentially mole for mole; negligible quantities of carbon dioxide were evolved. When previously dialyzed against distilled water, this system, upon incubation with homogentisic acid, gave rise to a ,&keto acid which decarboxylated much more slowly than acetoacetic acid in the presence of aniline citrate. As the rate of decarboxylation of P-keto acids in the presence of aniline citrate varies inversely with the length of the carbon chain (17), it appeared that a P-keto acid of longer chain length than acetoacetic acid had been formed in this dialyzed system. The biological activity of the long chain P-keto acid was indicated by its conversion, when incubated with an undialyzed preparation, to a p-keto acid which, on decarboxyla-