Direct Correlation between Donor-Acceptor Distance and Temperature Dependence of Kinetic Isotope Effects in Hydride-Tunneling Reactions of NADH/NAD Analogues.

Recent study of structural effects on primary kinetic isotope effects (1° KIEs) of H-transfer reactions in enzymes and solution revealed that a more rigid reaction system gave rise to a weaker temperature dependence of 1° KIEs, i.e., a smaller isotopic activation energy difference (Δ = - ). This has been explained within the contemporary vibrationally assisted activated H-tunneling (VA-AHT) model in which rigidity is defined according to the density of donor-acceptor distance (DAD) populations at the tunneling ready state (TRS) sampled by heavy atom motions. To test the relationship between DAD and Δ in the model, we developed a computational method to obtain the TRS structures for H-transfer reactions. The method was applied to three hydride transfer reactions of NADH/NAD analogues for which the Δ's as well as secondary (2°) KIEs have been reported. The 2° KIEs computed from each TRS structure were fitted to the observed values to obtain the optimal TRSs/DAD's. It was found that a shorter DAD does correspond with a smaller Δ. This appears to support the VA-AHT model. Moreover, an analysis of hybridizations at the bent TRS structures shows that rehybridizations at the donor-acceptor centers are much more advanced than predicted from the classical mechanism. This implies that more orbital preparations are required for the nonclassical H-tunneling to take place.