Purification and Characterization of Maleate Hydratase from Pseudomonas pseudoalcaligenes.

Maleate hydratase (malease) from Pseudomonas pseudoalcaligenes has been purified. The purified enzyme (98% pure) catalyzes the stereospecific addition of water to maleate and citraconate (2-methylmaleate), forming d-(+)-malate and d-(+)-citramalate, respectively. 2,3-Dimethylmaleate was also a substrate for malease. The stability of the enzyme was dependent on the protein concentration and the addition of dicarboxylic acids. The purified enzyme (89 kDa) consisted of two subunits (57 and 24 kDa). No cofactor was required for full activity of this colorless 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. Malease 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 constants for the maleate, citraconate, and 2,3-dimethylmaleate hydration reactions were 2,050, 104, and 11.2, respectively.