Studies on the mechanism of fatty acid synthesis: XXVI. Purification and properties of malonyl-coenzyme A—Acyl carrier protein transacylase of Escherichia coli

Abstract

Malonyltransacylase, purified 950-fold from extracts of Escherichia coli, was found to he homogeneous by disc-gel electrophoresis. The molecular weight of the enzyme was estimated to be 35,500 daltons, based upon polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The enzyme, while insensitive to sulfhydryl inhibitors, is strongly inactivated by phenylmethanesulfonylfluoride, suggesting that it possesses an active serine residue. The transacylase accepts the malonyl group from malonyl-CoA or malonyl-ACP to form a malonyl-enzyme intermediate. This intermediate is stable to performic acid oxidation; on hydrolysis with pepsin it yields peptic peptides also stable to performic acid. The malonyl group from malonyl-enzyme intermediate can be transferred to ACP, CoA, pantetheine, N-( N-acetyl-β-alanyl) cysteamine, or N-acetylcysteamine. The enzyme is specific for the malonyl group and does not transfer acetyl group from acetyl-CoA. But acetyl-CoA is a competitive inhibitor of malonyl-CoA, with a K i , value of 115 μ m. Data from kinetic studies of the transacylase reaction are consistent with the formation of a malonyl-enzyme intermediate during the reaction. Lineweaver-Burk plots at different concentrations of malonyl-CoA and at various fixed concentrations of ACP give a series of parallel lines. Moreover, CoA is found to be a competitive inhibitor of ACP, and malonyl-ACP a competitive inhibitor of malonyl-CoA. These findings are consistent with a Ping-Pong Bi Bi kinetic scheme for the malonyltransacylase reaction.