Solvent-Slaved Motions in the Hydride Tunneling Reaction Catalyzed by Human Glycolate Oxidase

Enzyme motions facilitate many hydride-transfer reactions involving quantum mechanical (QM) tunneling. The evidence mainly comes from the determination of kinetic isotope effects (KIEs) and their temperature dependence that have been used to reveal interesting characteristics of human glycolate oxidase ( Hs GOX). Previous studies have shown that Hs GOX oxidizes glycolate to glyoxylate via a hydride-transfer mechanism to an enzyme-associated FMN. Here, we investigate the temperature effect on the anaerobic rate of flavin reduction ( k red ) for Hs GOX with glycolate and [2 R - 2 H]glycolate. While the k red values for Hs GOX are temperature-dependent, their KIEs on the k red values ( D k red ) do not change as the temperature is varied. This is consistent with the involvement of QM hydride tunneling in the highly optimized active site of Hs GOX. We show that the enzyme motions are slaved by the fluctuations in the bulk solvent after determining the k red and D k red for Hs GOX at various solvent viscosities and constant temperature. These results are interpreted in the context of an extension of the Transition State Theory (TST) previously described for adiabatic processes. These experiments demonstrate for the first time that the solvent viscosity modulates the rate of hydride transfer in an enzyme-catalyzed reaction. Furthermore, molecular dynamics simulations show that an increase in the collision frequency of only the bulk solvent from 0.8 ps –1 to 3.8 ps –1 slows down the dynamics of the Hs GOX-glycolate complex in the active site, suggesting a direct coupling between solvent motions and the active site dynamics.