Origin of Enzymatic Kinetic Isotope Effects in Human Purine Nucleoside Phosphorylase

Here we report a study of the effect of heavy isotope labeling on the reaction catalyzed by human purine nucleoside phosphorylase (hPNP) to elucidate the origin of its catalytic effect and of the enzymatic kinetic isotope effect (EKIE). Using quantum mechanical and molecular mechanical (QM/MM) molecular dynamics (MD) simulations, we study the mechanism of the hPNP enzyme and the dynamic effects by means of the calculation of the recrossing transmission coefficient. A free energy surface (FES), as a function of both a chemical and an environmental coordinate, is obtained to show the role of the environment on the chemical reaction. Analysis of reactive and nonreactive trajectories allows us to study the geometric, dynamic, and electronic changes of the chemical system. Special attention is paid to the electrostatic potential created by the environment on those atoms involved in the chemical reaction. Some amino acid residues and solvent molecules that interact with the chemical system provide a specific configuration that electrostatically favor the reaction progress, producing a reactive trajectory. The EKIE is calculated within the framework of the variational transition state theory (VTST), giving very good agreement with the experimental data. According to our simulations the chemical reaction is slightly slower in the heavy enzyme than in its light counterpart enzyme because protein motions coupled to the reaction coordinate are slower. Thus, protein dynamics have a small but measurable effect on the chemical reaction rate