Probing the Mechanism of Inosine Monophosphate Dehydrogenase with Kinetic Isotope Effects and NMR Determination of the Hydride Transfer Stereospecificity

The mechanism of human type II inosine monophosphate dehydrogenase has been probed by measurements of primary deuterium kinetic isotope effects, and by determination of the stereochemical course of the reaction. The deuterium isotope effects onVmaxfrom [2-deutero]IMP are unity for reactions with a variety of monovalent cation activators (K+, NH4+, Na+, Rb+) of various efficacy. In each case normal effects onVmax/Kmin the range of 1.9 to 3.5 are observed for both IMP and NAD, and are larger for NAD. These results demonstrate that both substrates can dissociate from the E·M+·IMP·NAD complex, therefore the kinetic mechanism is not ordered as previous steady-state kinetic studies have suggested. Comparison of reaction rates in D2O and H2O show no2H isotope effect onVmax, and a ≤twofold decrease inVmax/Km; thus, a proton transfer from solvent is not rate-limiting in turnover. The NMR spectrum of the [4-deutero]NADH produced in the reaction of [2-deutero]IMP and NAD shows that the hydrogen is transferred to the B, or pro-S, side of the nicotinamide ring. Presteady-state kinetic experiments reveal a burst of NADH formation in the first turnover, demonstrating that a late step in the mechanism is rate-limiting. The rate of the burst phase is reduced ∼twofold with [2-deutero]IMP as substrate, indicating that the hydride transfer step is kinetically significant early in the reaction.