Oxaloacetate decarboxylase from Pseudomonas stutzeri: purification and characterization.
Oxaloacetate decarboxylase (OXAD), the enzyme that catalyzes the decarboxylation 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 chromatography, and biomimetic-dye affinity chromatography. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native high-performance gel-filtration liquid chromatography were, respectively, 63 and 64 kDa, suggesting a monomeric protein. OXAD required for maximum activity divalent metal cations such as Mn2+ and Mg2+ but not monovalent cations. The enzyme is not inhibited by avidin, but is competitively inhibited by adenosine 5'-diphosphate, acetic acid, phosphoenolpyruvate, malic acid, and oxalic acid. Initial velocity, product inhibition, and dead-end inhibition studies suggested a rapid-equilibrium ordered kinetic mechanism with Mn2+ being added to the enzyme first followed by oxaloacetate, and carbon dioxide is released first followed by pyruvate. Inhibition data as well as pH-dependence profiles and kinetic parameters are reported and discussed in terms of the mechanism operating for oxaloacetate decarboxylation.
Labrou
N E
NE
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, Athens, GR-11855-, Greece.
Clonis
Y D
YD
D001639:Bicarbonates / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019216:Metals, Heavy / Q000494:pharmacology; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*
D001639:Bicarbonates; D002413:Cations, Divalent; D019216:Metals, Heavy; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D002118:Calcium