Characterization of a membrane-bound biotin-containing enzyme: oxaloacetate decarboxylase from Klebsiella aerogenes.
Oxaloacetate decarboxylase from Klebsiella aerogenes is firmly bound to the cytoplasmic membrane, from which it can be solubilized with nonionic detergents. The solubilized enzyme behaved like the membrane-bound enzyme with respect to its inhibition by avidin and to the requirement of sodium ions for catalytic activity. The decarboxylase was purified 4.5-fold over the solubilized membrane extract by conventional means. Dodecyl-sulfate disc-gel electrophoretic analysis indicated that the enzyme consists of polypeptides of a single size. The molecular weight of these polypeptides is 68000. Radioactive biotin was incorporated specifically into these polypeptide chains upon growth of the bacteria in the presence of the radioactive vitamin. Biotin as the prosthetic group of oxaloacetate decarboxylase is now firmly established. The enzyme in the absence of detergent occurs in a highly aggregated form which elutes in the exclusion volume of a Biogel A 1.5 m column. The reported inhibition of oxaloacetate decarboxylase by citrate could not be repeated. On the other hand oxalate, 2-oxomalonate and glyoxylate proved to be very potent inhibitors of the decarboxylase. The stereochemical course of the oxaloacetate decarboxylation reaction was determined starting from stereospecifically labelled malates, which by malate dehydrogenase and oxaloacetate decarboxylase were converted to chiral pyruvates. The chirality of these pyruvates was analysed via their conversion to acetates and malates by determining the extent of tritium retention upon incubation of the latter with fumarase. It was found that oxaloacetate decarboxylation occurs stereospecifically with retention of configuration.
D001710:Biotin / Q000032:analysis*; D002262:Carboxy-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008293:Malates; D008970:Molecular Weight; D010071:Oxaloacetates / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D008293:Malates; D010071:Oxaloacetates; D001710:Biotin; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase