Hydrogen Tunneling, Electrostatics, and Conformational Motions in Enzyme Catalysis

Abstract

The roles of hydrogen tunneling, electrostatics, and conformational motions in enzyme catalysis will be discussed. We have developed hybrid quantum/classical molecular dynamics methods that include the quantum mechanical effects of the active electrons and transferring proton(s), as well as the motions of the entire solvated enzyme. These methods have been used to study the proton and hydride transfer reactions catalyzed by the enzymes dihydrofolate reductase (DHFR) and ketosteroid isomerase (KSI). The free energy profiles are generated along a collective reaction coordinate, and the changes in hydrogen bonding and electrostatic interactions are analyzed along the entire reaction pathway. An analysis of the simulations resulted in the identification and characterization of a network of coupled motions that extends throughout the enzyme and represents equilibrium conformational changes that facilitate the chemical reaction. Mutations distal to the active site are shown to significantly impact the catalytic rate constant by altering the conformational sampling of the entire enzyme, thereby changing the probability of sampling configurations conducive to the catalyzed reaction. We have also developed quantum mechanical/molecular mechanical methodology to calculate the vibrational frequency shifts of thiocyanate probes incorporated into the active site of an enzyme. This methodology is shown to reproduce the experimentally measured vibrational shifts upon binding of an intermediate analog to KSI for two different nitrile probe positions. Analysis of the simulations provides atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by the nitrile probe. This approach is also being used to study the vibrational shifts of nitrile probes for intermediates along the reaction pathway of DHFR to elucidate the conformational changes and the variation of the electrostatic microenvironments during catalysis.