Hydride-exchange reactions between NADH and NAD+ model compounds under non-steady-state conditions. Apparent and real kinetic isotope effects

Abstract

The kinetics of the hydride exchange reaction between NADH model compound 10-methyl-9,10-dihydroacridine (MAH) and 1-benzyl-3-cyanoquinolinium (BQCN⁺) ion in acetonitrile were studied at temperatures ranging from 291 to 325 K. The extent of reaction–time profiles during the first half-lives are compared with theoretical data for the simple single-step mechanism and a 2-step mechanism involving initial donor/acceptor complex formation followed by unimolecular hydride transfer. The profiles for the reactions of MAH deviate significantly from those expected for the simple single-step mechanism with the deviation increasing with increasing temperature. The deviation from simple mechanism behavior is much less pronounced for the reactions of 10-methyl-9,10-dihydroacridine-10,10-d₂ (MAD) which gives rise to extent of reaction dependent apparent kinetic isotope effects (KIE_app). Excellent fits of the experimental extent of reaction–time profiles with theoretical data for the 2-step mechanism, in the pre-steady-state time period, were observed in all cases. Resolution of the kinetics of the hydride exchange reaction into the microscopic rate constants over the entire temperature range resulted in real kinetic isotope effects for the hydride transfer step ranging from 40 (291 K) to 8.2 (325 K). That the reaction involves significant hydride tunnelling was verified by the magnitudes of the Arrhenius parameters; E_a^D − E_a^H = 8.7 kcal mol⁻¹ and A^D/A^H = 8 × 10⁴. An electron donor acceptor complex (λ_max = 526 nm) was observed to be a reaction intermediate. Theoretical extent of reaction–time profile data are discussed for the case where a reaction intermediate is formed in a non-productive side equilibrium as compared to the case where it is a real intermediate on the reaction coordinate between reactants and products. The common assumption that the two cases are kinetically indistinguishable is shown to be incorrect.