Structure-Reactivity Effects on Intrinsic Primary Kinetic Isotope Effects for Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase.

Primary deuterium kinetic isotope effects (1°DKIE) on (kcat /KGA , M-1 s-1 ) for dianion (X2- ) activated hydride transfer from NADL to glycolaldehyde (GA) catalyzed by glycerol-3-phosphate dehydrogenase were determined over a 2100-fold range of enzyme reactivity: (X2- , 1°DKIE); FPO3 2- , 2.8 ± 0.1; HPO3 2- , 2.5 ± 0.1; SO4 2- , 2.8 ± 0.2; HOPO3 2- , 2.5 ± 0.1; S2 O3 2- , 2.9 ± 0.1; unactivated; 2.4 ± 0.2. Similar 1°DKIEs were determined for kcat . The observed 1°DKIEs are essentially independent of changes in enzyme reactivity with changing dianion activator. The results are consistent with (i) fast and reversible ligand binding; (ii) the conclusion that the observed 1°DKIEs are equal to the intrinsic 1°DKIE on hydride transfer from NADL to GA; (iii) similar intrinsic 1°DKIEs on GPDH-catalyzed reduction of the substrate pieces and the whole physiological substrate dihydroxyacetone phosphate. The ground-state binding interactions for different X2- are similar, but there are large differences in the transition state interactions for different X2- . The changes in transition state binding interactions are expressed as changes in kcat and are proposed to represent changes in stabilization of the active closed form of GPDH. The 1°DKIEs are much smaller than observed for enzyme-catalyzed hydrogen transfer that occurs mainly by quantum-mechanical tunneling.