Enzyme-Catalyzed Acylation of Homoserine:  Mechanistic Characterization of the Escherichia coli metA-Encoded Homoserine Transsuccinylase

• Published in: Biochemistry (Easton) Vol. 38; no. 43; pp. 14416 - 14423
• Main Authors: Born, Timothy L, Blanchard, John S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26-10-1999
• Subjects: 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; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; homoserine; Homoserine - metabolism; Homoserine O-Succinyltransferase; homoserine transsuccinylase; Hydrogen-Ion Concentration; Iodoacetamide - pharmacology; Kinetics; Methionine - biosynthesis; Solvents
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi991710o

The first unique step in bacterial and plant methionine biosynthesis involves the activation of the γ-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 ∼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.