Enzyme-catalyzed acylation of homoserine: Mechanistic characterization of the Escherichia coli metA-encoded homoserine transsuccinylase

The first unique step in bacterial and plant methionine biosynthesis involv es the activation of the gamma-hydroxyl of homoserine. In Escherichia coli, this activation is accomplished via a succinylation reaction catalyzed by homoserine transsuccinylase. The activity of this enzyme is closely regulat ed in vivo and therefore represents a critical control point for cell growt h and viability. We have cloned homoserine transsuccinylase from E. coli an d present the first detailed enzymatic study of this enzyme. Steady-state k inetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the succinyl group of succinyl-CoA is initially transfe rred to an enzyme nucleophile before subsequent transfer to homoserine to f orm the final product, O-succinylhomoserine. The maximal velocity, V/Ksucci nyl-CoA, and V/K-homoserine all exhibited a bell-shaped pH dependence with apparent pKs of 6.6 and similar to 7.9, The enzyme was inhibited by iodoace tamide in a pH-dependent manner, with an apparent pK of the group being ina ctivated of 6.4. This suggests the presence of an active site cysteine whic h forms a succinyl-cysteine intermediate during enzymatic turnover, Solvent kinetic isotope effect studies yielded inverse effects of 0.7 on V and 0.6 1 on V/K in the reverse reaction only. On the basis of these observations, we propose a detailed chemical mechanism for this important member of the a cyltransferase family.