PURIFICATION AND CHARACTERIZATION OF MALEATE HYDRATASE FROM PSEUDOMONAS-PSEUDOALCALIGENES

Maleate hydratase (malease) from Pseudomonas pseudoalcaligenes has bee n purified. The purified enzyme (98% pure) catalyzes the stereospecifi c addition of water to maleate and citraconate (2-methylmaleate), form ing D-(+)-malate and D-(+)-citramalate, respectively. 2,3-Dimethylmale ate was also a substrate for malease. The stability of the enzyme was dependent on the protein concentration and the addition of dicarboxyli c acids. The purified enzyme (89 kDa) consisted of two subunits (57 an d 24 kDa). No cofactor was required for full activity of this colorles s enzyme. Maximum enzyme activity was measured at pH 8 and 45-degrees- C. The K(m) for maleate was 0.35 mM, and that for citraconate was 0.20 mM. Thiol reagents, such as p-chloromercuribenzoate and iodoacetamide , and sodium dodecyl sulfate completely inhibited malease activity. Ma lease activity was competitively inhibited by D-malate (K(i) = 0.63 mM ) and D-Citramalate (K(i) = 0.083 mM) and by the substrate analog 2,2- dimethylsuccinate (K(i) = 0.025 mM). The apparent equilibrium constant s for the maleate, citraconate, and 2,3-dimethyimaleate hydration reac tions were 2,050, 104, and 11.2, respectively.