Human Erythrocyte Glutathione Reductase: Chemical Mechanism and Structure of the Transition State for Hydride Transfer

Kinetic parameters and primary deuterium kinetic isotope effects for NADH and five pyridine nucleotide substrates have been determined at pH 8.1 for human erythrocyte glutathione reductase. ^DV/K_NADH and ^DV are equal to 1.4 and are pH independent below pH 8.1, but ^DV decreases to 1.0 at high pH as a group exhibiting a pK of 8.6 is deprotonated. This result suggests that as His-467′ is deprotonated, the rate of the isotopically insensitive oxidative half-reaction is specifically decreased and becomes rate-limiting. For all substrates, equivalent V and V/K primary deuterium kinetic isotope effects are observed at pH values below 8.1. The primary deuterium kinetic isotope effect on V, but not V/K, is sensitive to solvent isotopic composition. The primary tritium kinetic isotope effects agree well with the corresponding value calculated from the primary deuterium kinetic isotope effects by using the Swain-Schaad relationship. This suggests that the primary deuterium kinetic isotope effects observed in these steady-state experiments are the intrinsic primary deuterium kinetic isotope effects for hydride transfer. The magnitude of the primary deuterium kinetic isotope effect is dependent on the redox potential of the pyridine nucleotide substrate used, varying from ~ 1.4 for NADH and -320 mV reductants to 2.7 for thioNADH to 4.2-4.8 for 3-acetylpyridine adenine dinucleotide (3APADH). The α-secondary tritium kinetic isotope effects also increase as the redox potential of the pyridine nucleotide substrate becomes more positive. Together, these data indicate that the transition state for hydride transfer is very early for NADH and becomes later for thioNADH and 3APADH, as predicted by Hammond's postulate. The temperature dependence of the kinetic parameters and the primary deuterium kinetic isotope effects have been determined for NADH, thioNADH, and 3APADH. The energies of activation increase as the redox potential of the substrate becomes more positive. In all cases, the isotope effect on the energies of activation and the Arrhenius preexponential factors are close to unity. These data suggest that quantum mechanical tunneling is not significant in the hydride transfer reaction. A chemical mechanism for the reductive half-reaction of human erythrocyte glutathione reductase is proposed on the basis of these data.