Comparison of the Structures of Enzymatic and Nonenzymatic Transition States. Reductive Desulfonation of 4-X-2,6-Dinitrobenzenesulfonates by Reduced Nicotinamide Adenine Dinucleotide

Transition state structures for enzymatic and nonenzymatic direct hydride transfer reductions of 4-X-2.6-dinitrobenzenesulfonates by NADH are compared using two experimental approaches. These are (1) electronic substituent effects, giving information about the transfer of charge, and (2) protium-deuterium isotope effects giving information about the transfer of the hydrogen nucleus. Hammett plots for the nonenzymatic reaction (ρ = 4.97 using σ constants) have been reported previously (Kurz, L. C., and Frieden, C. (1975), J. Am. Chem. Soc. 97, 677) and it was concluded that considerable negative charge has been transferred from reductant to oxidant in the transition state. A lower limit of 11 on the ρ value for the equilibrium constant is now reported. Thus the extent of charge transfer in the transition state is less than 0.5 and it is not likely that the electron is transferred in a prior equilibrium step. For the enzymatic reaction, no significant correlation between Michaelis constants and σ or σ^- is found while the substituent dependence of the maximal velocities, V_max, is precisely the same as that found for the nonenzymatic second-order rate constants, k_N. Log-log plots of V_max vs. k_N have a slope of 1.01 ± 0.06. The primary isotope effect on ks is large, ∼4.7, while the secondary effect is normal, ∼1.2. It is concluded that the hydrogen nucleus is in flight in the transition state. Furthermore, with consideration of the electronic substituent effects, the electron and hydrogen nucleus transfers are closely coupled in these reactions. For the enzymatic reaction no isotope effects on Michaelis constants are found, while those on V_max are the same as those found for the nonenzymatic reaction. Thus the enzymatic reaction proceeds with a mechanism of prior equilibrium binding of both substrates followed by rate-determining hydride transfer. From these data, we conclude: (a) The structures of nonenzymatic and enzymatic transition states are quantitatively similar, (b) In these activated complexes the transfers of negative charge and the hydrogen nucleus are nearly synchronous.