Free Energy Simulations of Active-Site Mutants of Dihydrofolate Reductase

This study employs hybrid quantum mechanics molecular-mechanics (QM/MM) simulations to investigate the effect of mutations of the active-site residue 114 of E. coli dihydrofolate reductase (DHFR) on the hydride transfer. Recent kinetic measurements of the 114X mutants (X = V; A, and G) indicated slower hydride transfer fates and increasingly temperature-dependent kinetic isotope effects (KlEs) withsystematic reduction of the 114 side chain. The QM/MM simulations snow that when the original isoleucine residue is substituted in silico by valine, alanine, or glycine (I14V, I14A, and I14G DHFR, respectively), the free energy barrier- height of the hydride transfer, reaction increases relative to the wild-type-enzyme. These trends are in line with the single-turnover rate measurements reported for these systems. In addition, extended dynamics simulations of the reactive Michaelis complex reveal enhanced flexibility in the mutants, and in particular for the I14G mutant, including considerable fluctuations of the don-Or acceptor distance (DAD) and the active-sitehydrogen bonding network compared with those detected in the native enzyme. These 'observations suggest that the perturbations induced by the mutations partly impair the actiye-site environment in the reactant state. On the other hand, the average DADs at the transition state of all DHER variants are similar. Crystal structures of 114 Mutants,(V, A, and confirmed the trend of increased flexibility of the M20 and other loops.