PMID:10572016
| Citation |
Born, TL and Blanchard, JS (1999) Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Escherichia coli metA-encoded homoserine transsuccinylase. Biochemistry 38:14416-23 |
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| Abstract |
The first unique step in bacterial and plant methionine biosynthesis involves 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 regulated in vivo and therefore represents a critical control point for cell growth and viability. We have cloned homoserine transsuccinylase from E. coli and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the succinyl group of succinyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-succinylhomoserine. The maximal velocity, V/K(succinyl)(-)(CoA), and V/K(homoserine) all exhibited a bell-shaped pH dependence with apparent pK's of 6.6 and approximately 7.9. The enzyme was inhibited by iodoacetamide in a pH-dependent manner, with an apparent pK of the group being inactivated of 6.4. This suggests the presence of an active site cysteine which forms a succinyl-cysteine intermediate during enzymatic turnover. Solvent kinetic isotope effect studies yielded inverse effects of 0.7 on V and 0.61 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 acyltransferase family. |
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| Keywords |
Acylation; Acyltransferases/biosynthesis; Acyltransferases/genetics; Acyltransferases/isolation & purification; Acyltransferases/metabolism; Binding Sites; Catalysis; Cloning, Molecular; Cysteine/metabolism; Deuterium Oxide; Enzyme Activation/drug effects; Enzyme Inhibitors/pharmacology; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Homoserine/metabolism; Homoserine O-Succinyltransferase; Hydrogen-Ion Concentration; Iodoacetamide/pharmacology; Kinetics; Methionine/biosynthesis; Solvents |
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