Preferred order random kinetic mechanism for homoserine dehydrogenase of Escherichia coli (Thr-sensitive) aspartokinase/homoserine dehydrogenase-I: equilibrium isotope exchange kinetics.

Isotope exchange kinetics at chemical equilibrium have been used to investigate the kinetic mechanism of homoserine dehydrogenase (EC 1.1.1.3) of the (Thr-sensitive) aspartokinase/homoserine dehydrogenase-I multifunctional enzyme from E. coli. For the reaction (L-ASA + NADPH + H+ = L-Hse + NADP+), at pH 9.0, 37 degrees C, Keq = 100 (+/- 20). Under these conditions, the rate for exchange of 14C]-L-homoserine (Hse) in equilibrium L-aspartate-beta-semialdehyde (ASA) is nearly twice that for the 3H]-NADP+ in equilibrium NADPH exchange. This indicates that covalent interconversion between reactants and products bound in the active site cannot be rate-limiting. Upon variation of the concentrations of all four substrates in constant ratio at equilibrium (to minimize dead-end complex formation), the Hse in equilibrium ASA exchange increased smoothly toward a maximum. In contrast, the NADP+ in equilibrium NADPH exchange rate increased to a maximum value at partial saturation, then decreased to approximately half the maximum rate. These data are consistent with a preferred-order random kinetic mechanism in which the dominant pathway involves association of NADPH prior to L-ASA and dissociation of L-Hse prior to NADP+.