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

Abstract

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); FPO32–, 2.8 ± 0.1; HPO32–, 2.5 ± 0.1; SO42–, 2.8 ± 0.2; HOPO32–, 2.5 ± 0.1; S2O32–, 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.