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 (KIEapp). 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; Ea D - EaH = 8.7 kcal mol-1 and AD/AH = 8 x 10(4). An electron donor acceptor complex (lambda 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.

Guidelines for tunneling in enzymes

“…Although it is commonly believed that NADH is exclusively a two-electron, hydride transfer agent, there are clear indications of electron and proton transfer with obligate one-electron redox centers that do not involve transfer of a hydride [82]. Excellent examples are provided by ferricyanide [83] and ferrocenium ions [81, 84].…”

Intramolecular kinetic isotope effect in hydride transfer from dihydroacridine to a quinolinium ion. Rejection of a proposed two-step mechanism with a kinetically significant intermediate

“…14 Similarly, Table 1 lists rate constants and KIEs calculated according to simple second-order kinetics [eqn (1)], as obtained by Parker and coworkers. 15 Table 2 lists Parker's rate constants and KIEs, derived from fitting to the two-step kinetic scheme of eqn (2). The KIEs in Table 1 are quite ordinary, and in agreement with Kreevoy's, but those in Table 2 are unusually large, especially at lower temperatures.…”

“…Parker measured only the intermolecular KIE, as in Table 1. 15 His evidence for a kinetically significant intermediate was the inadequacy of the fit of the data to simple second-order kinetics. Thus the KIEs in Table 2 were not measured directly, but only by adjusting the observed KIEs for the kinetic complexity of eqn (2).…”

“…The reaction is indeed reversible, with an equilibrium constant of only 6.7 or 11.7 favoring products. 14, 15 Reversibility would then equilibrate H and D among all species and increase the D content of 3. Indeed, when the reaction was allowed to proceed for 24 h, the ratio of [3-h 2 ] to [3-d] was found to be 1.12, corresponding to an apparent KIE near 1.…”

Nonconventional versus conventional application of pseudo‐first‐order kinetics to fundamental organic reactions

“…We presented an alternative three-step mechanism in 2003 [15], which was challenged by Perrin and Zhao in 2008 [16]. The mechanism has been found to be even more complex than we first believed [15] and was shown to involve an oxygen-catalyzed chain reaction even when carried out in a glove box under a nitrogen atmosphere [7].…”