Kinetics and catalytic properties of coenzyme A transferase from Peptostreptococcus elsdenii.
Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii was purified to homogeneity, and some of its physical and catalytic properties were determined. The native enzyme has a molecular weight of 181,000 and is composed of two alpha subunits (molecular weight, 65,000) and one beta subunit (molecular weight 50,000). Heat treatment of the crude cell extract to 58 degrees C causes proteolysis of the native enzyme and yields a catalytically active enzyme with an approximate molecular weight of 120,000. The native CoA transferase is specific for CoA esters of short-chain alkyl monocarboxylic acids. With acetate as CoA acceptor the enzyme is active with propionyl-, butyryl-, isobutyryl-, valeryl-, isovaleryl,- and hexanoyl-CoA but not with heptanoyl or longer-chain CoA esters. There is no activity with acetoacetyl-CoA or the CoA esters of dicarboxylic acids. Steady-state kinetics indicated that the reaction proceeds via a classical bi-, bi-ping-pong mechanism. Maximal activity is obtained with propionyl- or butyryl-CoA, and both the Vmax and Km decrease as the alkyl chain length of the CoA ester increases. All CoA esters apompetitive inhibitor although it is not active as a substrate. Evidence for an enzyme CoA intermediate was provided by demonstration of an exchange between 14C-free acids (acetate and butyrate) and their corresponding CoA esters and by isolation of a 3H-labeled CoA enzyme after incubation of the enzyme with 3H-labeled acetyl-CoA. Approximately 2 mol of CoA was bound per mol of enzyme.
D001665:Binding Sites; D002264:Carboxylic Acids / Q000378:metabolism; D002845:Chromatography; D003065:Coenzyme A; D003998:Dicarboxylic Acids / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008970:Molecular Weight; D010462:Peptostreptococcus / Q000201:enzymology*; D014166:Transferases / Q000302:isolation & purification / Q000378:metabolism*
D002264:Carboxylic Acids; D003998:Dicarboxylic Acids; D014166:Transferases; D003065:Coenzyme A