How Conformational Dynamics of DNA Polymerase Select Correct Substrates: Experiments and Simulations

Nearly every enzyme undergoes a significant change in structure after binding it's substrate. Experimental and theoretical analyses of the role of changes in HIV reverse transcriptase structure in selecting a correct substrate are presented. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile of the conformational change commensurate with experimental data. A large conformational change occurring on a millisecond timescale locks the correct nucleotide at the active site but promotes release of a mismatched nucleotide. The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, whereas the same steps diminish the reaction probability of an incorrect substrate. º Kinetics and atomic simulations quantitatively explain nucleotide binding to HIV-RT º Reaction path, free energy, and kinetics of HIV-RT transition are computed º Simulations show that specificity is based on induced fit º Specificity is determined by the rate of conformation change not only chemical step