Comparison of the structures of enzymatic and nonenzymatic transition states. Reductive desulfonation of 4-X-2,6-dinitrobenzenesulfonates by reduced nicotinamide adenine dinucleotide

Kurz, L.C.; Frieden, C.

Biochemistry 16(24): 5207-5216

1977


ISSN/ISBN: 0006-2960
PMID: 200257
DOI: 10.1021/bi00643a008
Accession: 068523805

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Abstract
Transition state structures for enzymatic and nonenzymatic direct hydride transfer reductions of 4-X-2,6-dinitrobenzenesulfonates by NADH are compared using 2 experimental approaches. These are electronic substituent effects, giving information about the transfer of charge, and protium-deuterium isotope effects giving information about the transfer of the hydrogen nucleus. Hammett plots for the nonenzymatic reaction (.rho. = 4.97 using .sigma. constants) were reported previously considerable negative charge was transferred from reductant to oxidant in the transition state. A lower limit of 11 on the .rho. 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 Km and .rho. or .sigma.- is found while the substituent dependence of the maximal velocities, Vmax, is precisely the same as that found for the nonenzymatic 2nd-order rate constants, kN. Log-log plots of Vmax vs. kN have a slope of 1.01 .+-. 0.06. The primary isotope effect on kN is large, .apprx. 4.7, while the secondary effect is normal, .apprx. 1.2. The hydrogen nucleus is probably in flight in the transition state. 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 Km are found, while those on Vmax 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. The structures of nonenzymatic and enzymatic transition states are probably quantitatively similar. In these activated complexes the transfers of negative charge and the hydrogen nucleus are probably nearly synchronous.