Enzymes Responsible for Degradation of 4-Oxalmesaconic Acid in Pseudomonas ochraceae

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Abstract
The enzyme responsible for the degradation of 4-oxalmesaconate was partially purified from Pseudomonas ochraceae grown with phthalate. Column chromatography on DEAE-cellulose caused separation into two distinct enzymes, I and II. 4-Oxalmesaconate was converted into pyruvate and oxalacetate in the presence of MgCl2 and enzymes I and II. Optimum pH of the reaction was observed at pH 8.2 in Tris-HCl buffer. MgCl2 could be replaced by MnCl2 or CoCl2. Both enzymes were stable to heat-treatment at 65°C for 10 min. Analyses of time course, products and substrate specificity of the enzyme reaction accounted for the functions of two enzymes. Enzyme I (molecular weight 55,000, isoelectric point 5.1) hydrated 4-oxalmesaconate to give 4-oxalcitramate and may be Classified as a hydro-lyase. Enzyme II (160,000, 5.0) catalyzed the aldolitic Cleavage of 4-oxalcitramalate to pyruvate and oxalacetate in the presence of MgCl2. Enzyme II also Cleaved 4-hydroxy-4-methyl-2-oxoglutarate into pyruvate. Stoichiometry of the enzyme reaction suggested that enzyme II-catalyzed Cleavage occurred on only one enan-tiomer of the substrates. Furthermore, the metabolic pathway for the dissimilation of protocatechuate in P. ochraceae is presented and discussed in comparison with the pathway postulated previously by other workers.