Fumarylacetoacetic acid

Ravdin and Crandall (695) isolated a protein fraction from rat liver which converted homogentisic acid to a jS-keto acid decarboxylated slowly by aniline citrate at 38°C. A second enzyme fraction was obtained which converted this keto acid to acetoacetic acid. The /3-keto acid was isolated as its silver salt and was found also to be a dicarboxylic acid and a /3-diketone, and to give fumaric and acetoacetic acids on hydrolysis. The proposed formulation as fumarylacetoacetic acid (see diagram 8) has since beeri amply confirmed. Conversion of homogentisic acid to fumarylacetoacetic acid by a liver preparation involves uptake of the expected two atoms of oxygen (e.g., 489, 523). 

The enzyme hydrolyzing fumarylacetoacetic acid to fumaric and aceto-acetic acids has as yet been little studied. It may be the same as acylpyru-vase (592) or the triacetic acid hydrolyzing enzyme (154). 

Fumarylacetoacetic acid hydrolase Fumaric acid + Acetoacetic acid... 

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. 

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. 

The nature of the reaction catalyzed by homogentisic oxidase was, not established immediately. Ravdin and Crandall (1951) isolated and identified fumarylacetoacetic acid as a product, but Knox and Edwards (1955b) subsequently found that the primary product is an isomer, maleylacetoacetic acid, that is converted enzymically to the compound of Ravdin and Crandall (Fig. 13). The formation of maleylacetoacetate... 

The gene mutation inhibits hydrolytic cleavage of fumarylacetoacetate into fumarate and acetoacetate. Consequently, the toxic precursors maleylacetoacetate and fumarylacetoacetate accumulate in the liver and kidneys. They possess a reactive double bond and can therefore react with macromolecules to assume the properties of alkylating substances. In addition, intracellular glutathione deficiency develops due to the stable complex formation with glutathione, favouring lipid peroxidations. Enhanced formation of 5-aminolaevulinic acid can also be observed during occasional attacks of acute intermittent porphyria (G. Mitchel et al., 1990). 

Conversion of Homogentisic Add lo Maleylacetoacetic, Fumarylacetoacetic, Fumaric, and Acetoacetic Acids... 

This compound differs slightly from the isomeric fumarylacetoacetate in its absorption spectrum near 300 mu at neutral and alkaline pH values, but the maleyl compound has essentially no absorption at pH 1 whereas the fumaryl compound has a strong peak at 310 miu in acid. Fumarylacetoacetate had previously been isolated as a product of homogentisic oxidation by Ravdin and Crandall. Knox and collaborators have shown that the fumaryl compound is formed secondarily by a cis-trans isomerase, an enzyme that requires glutathione for activity and has in addition essential sulfhydryl groups on the protein. The isomerization has not been reversed. 

Fumarylacetoacetate is split to fumarate and acetoacetate by an enzyme that was known previously to hydrolyze diketo acids. It has been called acylpyruvase, triacetic acid hydrolyzing enzyme, and /J-diketonase. Since both the rate of hydrolysis of fumarylacetoacetate and its affinity for the enzyme exceed those of other substrates there is some justification for the name fumarylacetoacetate hydrolase. The irreversible action of this enzyme results in the formation of products that are metabolized by the systems previously described for fatty acid oxidation and the Krebs cycle. 

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. 

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