Oxaloacetate decarboxylase from Pseudomonas stutzeri: Purification and characterization

Oxaloacetate decarboxylase (OXAD), the enzyme that catalyzes the decarboxyl ation of oxaloacetate to pyruvic acid and carbon dioxide, was purified 245- fold to homogeneity from Pseudomonas stutzeri. The three-step purification procedure comprised anion-exchange chromatography, metal-chelate affinity c hromatography, and biomimetic-dye affinity chromatography. Estimates of mol ecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native high-performance gel-filtration liquid chromatography were, resp ectively, 63 and 64 kDa, suggesting a monomeric protein. OXAD required for maximum activity divalent metal cations such as Mn2+ and Mg2+ but not monov alent cations. The enzyme is not inhibited by avidin, but is competitively inhibited by adenosine 5'-diphosphate, acetic acid, phosphoenolpyruvate, ma lic acid, and oxalic acid. Initial velocity, product inhibition, and dead-e nd inhibition studies suggested a rapid-equilibrium ordered kinetic mechani sm with Mn2+ being added to the enzyme first followed by oxaloacetate, and carbon dioxide is released first followed by pyruvate. Inhibition data as w ell as pH-dependence profiles and kinetic parameters are reported and discu ssed in terms of the mechanism operating for oxaloacetate decarboxylation. (C) 1999 Academic Press.