The overall objective of these projects is to illuminate the role of hydrogen bonding, hydrogen tunneling, electrostatics, and conformational motions, as well as the impact of distal mutations, in enzyme reactions. Movies of hydrogen tunneling in LADH and DHFR are available below.
Our simulations suggested the concept of a free energy landscape for enzyme catalysis and the significant role of equilibrium, thermal conformational motions that lead to sampling of configurations conducive to chemistry.
We have developed a hybrid quantum/classical molecular dynamics approach for simulating proton and hydride transfer reactions in enzymes.41, 46, 48 This hybrid approach includes electronic and nuclear quantum effects, as well as the motion of the entire solvated enzyme. The methodology provides detailed mechanistic information at the molecular level and allows the calculation of rate constants and kinetic isotope effects. It also enables us to investigate the relation between enzyme motion and activity. We have also developed computational methodology for calculating the vibrational shifts of nitrile probes (i.e., the vibrational Stark effect) upon ligand binding and along catalytic cycles in enzymes to investigate the role of electrostatics in enzyme catalysis.159, 179, 190
We have applied this hybrid approach to hydride transfer in liver alcohol dehydrogenase (LADH),34, 37, 41, 48 dihydrofolate reductase (DHFR),50, 51, 56, 63, 67, 71, 80, 83, 84, 113, 165, 179, 185 and ketosteroid isomerase (KSI)121, 127, 143, 159 These simulations have illuminated the roles of hydrogen bonding, hydrogen tunneling, electrostatics, and conformational motions, as well as the impact of distal mutations.
We have used quantum mechanical/molecular mechanical (QM/MM) free energy simulation methods, as well as other computational approaches, to study the self-cleavage mechanism of the HDV ribozyme.129, 137, 141, 150, 162, 170, 173, 191 Our calculations indicate that the self-cleavage reaction of the HDV ribozyme is concerted with a phosphorane-like transition state when a divalent ion, Mg2+ or Ca2+, is bound at the catalytic site but is sequential with a phosphorane intermediate when a monovalent ion, such as Na+, is at this site. These observations are consistent with available experimental data. We have performed similar types of studies for the glmS ribozyme188, 214, 222 and the twister ribozyme.210
A comparison of the minimum free energy paths and free energy surfaces for the reaction with two and three divalent metal ions in the active site elucidated the possible role of the third metal ion. For the two-metal system, the minimum free energy path corresponds to a sequential mechanism, in which the phosphoryl transfer reaction is followed by relatively fast proton transfer. For the three-metal system, the minimum free energy path corresponds to only the phosphoryl transfer reaction without a subsequent proton transfer reaction. Our analysis indicates that the proton transfer reaction does not occur in the three-metal system because of electrostatic effects, where the positively charged metal ion partially neutralizes the negatively charged leaving group and thereby disfavors protonation. Moreover, these electrostatic interactions stabilize the product, making the phosphoryl transfer reaction more thermodynamically favorable with a lower free energy barrier relative to the activated state corresponding to the deprotonated 3’OH nucleophile.