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 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.
Born
T L
TL
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Blanchard
J S
JS
D000215:Acylation; D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D017666:Deuterium Oxide; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006714:Homoserine / Q000378:metabolism*; D051082:Homoserine O-Succinyltransferase; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008715:Methionine / Q000096:biosynthesis; D012997:Solvents
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D012997:Solvents; D006714:Homoserine; D008715:Methionine; D000217:Acyltransferases; D051082:Homoserine O-Succinyltransferase; C494973:metA protein, E coli; D017666:Deuterium Oxide; D003545:Cysteine; D007460:Iodoacetamide