title: Preferred order random kinetic mechanism for homoserine dehydrogenase of Escherichia coli (Thr-sensitive) aspartokinase/homoserine dehydrogenase-I: equilibrium isotope exchange kinetics.
abstract: 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+.
authors.affiliation: Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
authors.forename: F C
authors.initials: FC
authors.lastname: Wedler
authors.forename: B W
authors.initials: BW
authors.lastname: Ley
authors.forename: S L
authors.initials: SL
authors.lastname: Shames
authors.forename: S J
authors.initials: SJ
authors.lastname: Rembish
authors.forename: D L
authors.initials: DL
authors.lastname: Kushmaul
mesh_terms: D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006714:Homoserine / Q000378:metabolism; D006715:Homoserine Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009249:NADP / Q000378:metabolism
chemical_list: D001224:Aspartic Acid; C006644:aspartic semialdehyde; D009249:NADP; D006714:Homoserine; D006715:Homoserine Dehydrogenase