Kinetic and regulatory mechanisms for (Escherichia coli) homoserine dehydrogenase-I. Equilibrium isotope exchange kinetics.
Isotope exchange kinetics at chemical equilibrium were used to probe the mechanisms of substrate binding and regulatory behavior of homoserine dehydrogenase-I from Escherichia coli. At pH 9.0, 37 degrees C, Keq = 100 (+/- 20) for the catalyzed reaction: L-aspartate-beta-semialdehyde + NADPH + H+ = L-homoserine + NADP+. Saturation curves for the exchange reactions, [14C]L-homoserine <--> L-aspartate-beta-semialdehyde and [3H]NADP+ <--> NADPH were observed as a function of different reactant-product pairs, varied in constant ratio at equilibrium. The NADP+ <--> NADPH exchange rate was inhibited upon variation of pairs involving L-aspartate-beta-semialdehyde and L-homoserine, consistent with preferred order random binding of cofactors before amino acids. Optimal rate constants, derived by simulations of equilibrium isotope exchange kinetics data with the ISOBI program, indicate faster dissociation of amino acids than cofactors from the central complexes but nearly equal rates for association of cofactors and amino acids to free enzyme. Rate limitation of net turnover in both directions is determined by dissociation of cofactor from the E-cofactor complex. The allosteric modifier, L-threonine, produces distinctive perturbations of the saturation curves for isotope exchange, which were analyzed systematically with the ISOBI program. The best fit to the data was obtained by L-threonine inhibiting catalysis between the central complexes without altering substrate association-dissociation rates. Simulations also showed that rate-limiting catalysis suppresses the kinetic inhibition effects that are characteristic of preferred order substrate binding, producing patterns typical for a (rapid equilibrium) random kinetic scheme.
Wedler
F C
FC
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Ley
B W
BW
D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006715:Homoserine Dehydrogenase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D009249:NADP / Q000737:chemistry; D013912:Threonine / Q000737:chemistry
D007554:Isotopes; D013912:Threonine; D009249:NADP; D006715:Homoserine Dehydrogenase