Purification and kinetic properties of butyryl-CoA synthetase from Paecilomyces varioti.

Abstract

A butyryl-CoA synthetase (EC 6.2.1.2) was obtained from Paecilomyces varioti AHU 9417 in a highly purified form, which appeared to be homogeneous on polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate (SDS). The purified enzyme exhibited molecular weights of 145,000 for the native enzyme and 74,000 for the subunit. This enzyme required both monovalent and divalent cations for the reaction. The enzyme was active toward saturated fatty acids with 3 to 5 carbon atoms, and butyrate was the best substrate. The reaction mechanism of the enzyme was also investigated and was found to conform to a Bi-Uni-Uni-Bi ping-pong mechanism, in which the order of addition and evolution of substrates and products was ATP, butyrate, PPi, CoA, butyryl-CoA (or AMP) and AMP (or butyryl-CoA). Butyryl-CoA and AMP were evolved at random or at the same time.