Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) in the presence of NADPH. The key hydride transfer step in the reaction is facilitated by a combination of enzyme active site preorganization and correlated protein motions in the Michaelis-Menten (E:NADPH:DHF) complex.1 The present theoretical study employs mutagenesis to examine the relation between structural and functional properties of the enzyme. We mutate D122 in Escherichia coli DHFR, which is a highly conserved amino acid in the DHFR family.1 The consequent effect of the mutation on enzyme catalysis is examined from an energetic, structural and dynamic perspective. We employed a hybrid quantum-mechanical/molecular mechanical (QM/MM) approach with a recently developed semi-empirical AM1-SRP Hamiltonian that provides accurate results for this reaction.2-3 Our investigations suggest that the structural and dynamic perturbations caused by Asp122 mutations are along the reaction coordinate and lower the rate of hydride transfer. Importantly, analysis of the correlated and principle component motions in the enzyme suggest that the mutation alters the coupled motions that are present in the wild-type enzyme. Additionally, structural analyses from the present study show that mutation of Asp122 perturbs the local active site environment via lost interactions with the NADPH cofactor and Met20 loop residues, resulting in a change in the active site hydration.
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