Computer simulation of primary kinetic isotope effects in the proposed rate-limiting step of the glyoxalase I catalyzed reaction

The proposed rate-limiting step of the glyoxalase I catalyzed reaction is t he proton abstraction from the C1 carbon of the substrate by Glu(172). Here we examine primary kinetic isotope effects and the influence of quantum dy namics on this process by computer simulations. The calculations utilize th e empirical valence bond method in combination with the molecular dynamics free energy perturbation technique and path integral simulations. For the e nzyme-catalyzed reaction a H/D kinetic isotope effect of 5.0 +/- 1.3 is pre dicted in reasonable agreement with the experimental result of about 3. Fur thermore, the magnitude of quantum mechanical effects is found to be very s imilar for the enzyme reaction and the corresponding uncatalyzed process in solution, in agreement with other studies. The problems associated with at taining the required accuracy in order for the present approach to be usefu l as a diagnostic tool for the study of enzyme reactions are also discussed .