Fumarate from homogentisate

The pathway from homogentisate (7) to acetoacetate (9) and fumarate (10) proceeds by oxidative fission of the aromatic ring of homogentisate to give maleyl acetoacetate (8), which is then iso-merised to fumaryl acetoacetate . Hydrolysis of this intermediate then yields fumarate and acetoacetate S Figure 4.1. 

The 2-oxoacid p-hydroxyphenylpyruvate is decar-boxylated by the action of a dioxygenase (Eq. 18-49). The product homogentisate is acted on by a second dioxygenase, as indicated in Fig. 25-5, with eventual conversion to fumarate and acetoacetate. A rare metabolic defect in formation of homogentisate leads to tyrosinemia and excretion of hawkinsin97 a compound postulated to arise from an epoxide (arene oxide) intermediate (see Eq. 18-47) which is detoxified by a glutathione transferase (Box 11-B). 

Tyrosine, obtained from the diet or by hydroxylation of phenylalanine, is converted to homogentisate, whose aromatic ring is opened and cleaved, forming fumarate and acetoacetate (Figure 7-12). 

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). 

Figure 4. Chromatogram of a mixture of carboxylic acids as the t-butyidimethylsilyl derivatives. GC conditions DB-1 fused-silica capillary column (30 m x 0.32 mm i.d, 0.25 pm), initially at 60 "C for 2 min, then programmed to 280 °C at 4°C/min 0.8 pi sample, injected with split ratio of 15 1 both injector and detector temperatures at 300 °C nitrogen as the carrier gas at 0.9ml/min. Peaks l = formic, 2 = acetic, 3 = propionic, 4 = isobutyric, 5 = butyric, 6 — isovaleric, 7 = valeric, 8 = caproic. 9 = enanthic, 10 = benzoic, 11= caprylic, 12 = lactic, 13 = phenylacetic, 14 = glycollic, 15 = oxalic, 16 = pelargonic. 17 = malonic, 18 = capric, 19 - succinic, 20 - methylsuccinic, 21 = undecanoic, 22 = fumaric, 23 = 5-phenylvaleric, 24 = p-aminobenzoic, 25 = lauric. 26 = mandelic, 27 = adipic, 28 = 3-methyladipic, 29 = tridecanoic, 30 = phenyllactic. 31 = hippuric, 32 = myristic, 33 = p-hydroxybenzoic, 34 = malic, 35 = suberic, 36 = pentadecanoic, 37 = vanillic, 38 = palmitic. 39 = syiingic, 40 = tartaric, 41 — margaric, 42 = a-resorcylic, 43 = p-hydroxymandelic, 44 = y-resorcylic, 45 = stearic. 46 = homogentisic, 47 = protocatechuic 48 = nonadecanoic, 49 = citric 50 = cirachidic acid. (Reproduced from Ref. 299 with permission).

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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