Strategies toward a complete definition of an enzymatic reaction coordinate

Abstract

A theoretically based computational method has been developed in our group to identify protein motions, symmetrically coupled to the reaction coordinate, which modulate the width and height of the barrier to reaction. Previous studies have applied the method to horse liver alcohol dehydrogenase to help explain experimental kinetic isotope effects. In this work, the method has been applied to two isoforms of human lactate dehydrogenase (LDH) enzymes which facilitate hydride and proton transfer during the interconversion of pyruvate and lactate. LDH isoforms evolved to accommodate substrate demand in different parts of the body. The active sites of the isoforms are identical in amino acids content yet the kinetics are distinct. Molecular dynamics simulation were implemented for each isoform with either substrate bound. The signature of the Protein Promoting Vibration is distinct for each isoform due to differences in the donor-acceptor distance. We hypothesize that kinetic control of hydride transfer may be exerted via a decreased donor-acceptor distance when lactate is bound to the heart isoform and when pyruvate is bound to the muscle isoform. The identity and frequency of the active site residues correlated to the donor-acceptor motion vary for each isoform, demonstrating that differences remote from the active site affect reaction dynamics. The Transition Path Sampling (TPS) algorithm was applied to the heart isoform, establishing for the computational biology community the algorithm's efficiency for multidimensional systems whose energy surface is a complex terrain of valleys and saddle points. As a Monte Carlo importance sampling method, TPS is capable of surmounting barriers in path phase space, and focuses simulation on the rare event of enzyme catalyzed atom transfers. Generation of the transition path ensemble confirms both concerted and stepwise mechanisms for the catalyzed hydride and proton transfers. To identify a reduced reaction coordinate, time series of donor-acceptor distances and residue distances from the active site were examined. During the transition from pyruvate to lactate, residues located behind the transferring hydride compress towards the active site, causing residues located behind the hydride acceptor to relax away. It is demonstrated that an incomplete compression/relaxation transition across the donor-acceptor axis compromises the reaction.