Enzymatic Processes

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.

We have written several reviews on enzyme motion and catalysis.55, 58, 79, 81, 101, 131, 149171190

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.

Proton and hydride transfer reactions in enzymes

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.159179190

We have applied this hybrid approach to hydride transfer in liver alcohol dehydrogenase (LADH),34, 37, 4148 dihydrofolate reductase (DHFR),50, 51, 56636771808384113165179185 and ketosteroid isomerase (KSI)121, 127, 143159  These simulations have illuminated the roles of hydrogen bonding, hydrogen tunneling, electrostatics, and conformational motions, as well as the impact of distal mutations.

Movies of H tunneling in LADH and DHFR

Proton-coupled electron transfer in enzymes

We have developed a theoretical formulation for proton-coupled electron transfer (PCET) reactions in enzymes. This theory includes the quantum mechanical effects of the active electrons and the transferring proton, as well as the motions of all atoms in the complete solvated enzyme system. We have derived a series of nonadiabatic rate constant expressions that are valid in well-defined regimes. In this theory, the rate constant and kinetic isotope effect (KIE) are strongly influenced by the equilibrium proton donor-acceptor distance and frequency, the vibronic coupling, the reaction free energy, and the protein/solvent reorganization energy.  We have applied this theory to PCET in soybean lipoxygenase.6493126182213216219226

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Ribozymes

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, 141150, 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

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Human DNA Polymerase η

High-energy ultraviolet radiation damages DNA through the formation of cyclobutane pyrimidine dimers and leads to replication stalls. Human DNA polymerase η (Pol η) is an enzyme that rescues these stalled replication forks, in particular at thymine-thymine dimers (TTDs), by extending the primer with the correct complementary bases opposite the damaged site. In addition to the two Mg2+ions aligning the active site, experiments suggest that a third Mg2+ion could play an essential catalytic role. We used mixed quantum mechanical/molecular mechanical (QM/MM) free energy simulations to shed light on the role of this third metal ion.237

The specific method we used to study the catalytic role of the third active site divalent metal ion was a finite temperature string method with umbrella sampling in conjunction with a QM/MM treatment. We used this approach to generate the minimum free energy path and the relevant regions of the multidimensional free energy surface for the phosphoryl transfer reaction (Figure 1). We also investigated the possibility of proton transfer from the incoming nucleotide to the pyrophosphate leaving group.  Our simulations considered both sequential and concerted mechanisms for the phosphoryl transfer and proton transfer steps.

Figure 1.Proposed mechanism of Pol η.  An undetermined base B deprotonates O3′ of thymine, preparing it for phosphoryl transfer (red arrows in reactant). A possible self-activating deprotonation of the incoming nucleotide (adenine) was also studied (shown with question mark in product).  Coordination of the third metal to the leaving group is also shown in product.

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.