QM/MM simulations for methyl transfer in solution and catalysed by COMT: ensemble-averaging of kinetic isotope effects

Sampling of structures from QM/MM molecular dynamics reveals distinct families of reactant-state conformers and yields kinetic isotope effects for reactions in enzyme active sites and in solution, averaged over thermal fluctuations of the environment, that allows meaningful comparison of computed with experimental value

QM/MM kinetic isotope effects for chloromethane hydrolysis in water

Computational simulations for chloromethane hydrolysis have been performed using hybrid quantum-mechanical/molecular-mechanical methods with explicit solvation by large numbers of water molecules. In the first part of the paper, we present results for 2° 2H3, 1° 14C, and 1° 37Cl kinetic isotope effects (KIEs) at 298 K with both the AM1/TIP3P and B3LYP/6-31G* QM methods for the nucleophile H2O and electrophile CH3Cl surrounded by 496 solvating TIP3P water molecules. An initial Hessian computed for a subset of this system including up to 104 MM water molecules was reduced in size by successive deletion of rows and columns, and KIEs were evaluated for each. We suggest that accurate calculations of KIEs in solvated systems should involve a subset Hessian including the substrate together with any solvent atoms making specific interactions with any isotopically substituted atom. In the second part of the paper, the ensemble-averaged 2° α-2H3 KIE calculated with the B3LYP/6-31+G(d,p)/TIP3P method is shown to be in good agreement with experiment. This comparison is meaningful because it includes consideration of uncertainties owing to sampling of a range of representative thermally accessible solvent configurations. We also present ensemble-averaged 14C and 37Cl KIEs which have not as yet been determined experimentally. Copyright © 2013 John Wiley & Sons, Ltd.We thank the EPSRC for financial support (EP/E019455/1), the Juan de la Cierva subprogramme of the Spanish Ministry of Science (J.J.R.P.) and the General Secretariat of the Spanish Ministry for Education (I.H.W.) for financial support (SAB2010-0116) during sabbatical leave, Professors Iñaki Tuñón (Valencia) and Vicente Moliner (Castellon) for helpful discussions, and the University of Bath for access to its High Performance Computing Facility

Mechanism of glycoside hydrolysis:A comparative QM/MM molecular dynamics analysis for wild type and Y69F mutant retaining xylanases

Computational simulations have been performed using hybrid quantum-mechanical/ molecular-mechanical potentials to investigate the catalytic mechanism of the retaining endo-b-1, 4-xylanase (BCX) from B. circulans. Two-dimensional potential-of-mean-force calculations based upon molecular dynamics with the AM1/OPLS method for wild-type BCX with a p-nitrophenyl xylobioside substrate in water clearly indicates a stepwise mechanism for glycosylation: the rate-determining step is nucleophilic substitution by Glu78 to form the covalently bonded enzyme-substrate intermediate without protonation of the leaving group by Glu172. The geometrical configuration of the transition state for the enzymic reaction is essentially the same as found for a gas-phase model involving only the substrate and a propionate/propionic acid pair to represent the catalytic glutamate/glutamic acid groups. In addition to stabilizing the 2,5B boat conformation of the proximal xylose in the non-covalent reactant complex of the substrate with BCX, Tyr69 lowers the free-energy barrier for glycosylation by 42 kJ mol-1 relative to that calculated for the Y69F mutant, which lacks the oxygen atom OY. B3LYP/6-31+G* energy corrections reduce the absolute height of the barrier to reaction. In the oxacarbenium ion-like transition state OY approaches closer to the endocyclic oxygen Oring of the sugar ring but donates its hydrogen bond not to Oring but rather to the nucleophilic oxygen of Glu78. Comparison of the average atomic charge distributions for the wild-type and mutant indicates that charge separation along the bond between the anomeric carbon and Oring is matched in the former by a complementary separation of charge along the OY–HY bond, corresponding to a pair of roughly antiparallel bond dipoles, which is not present in the latte

Computational Treatment of Metalloproteins

Metalloproteins present a considerable challenge for modeling, especially when the starting point is far from thermodynamic equilibrium. Examples include formidable problems such as metalloprotein folding and structure prediction upon metal addition, removal, or even just replacement; metalloenzyme design, where stabilization of a transition state of the catalyzed reaction in the specific binding pocket around the metal needs to be achieved; docking to metal-containing sites and design of metalloenzyme inhibitors. Even more conservative computations, such as elucidations of the mechanisms and energetics of the reaction catalyzed by natural metalloenzymes, are often nontrivial. The reason is the vast span of time and length scales over which these proteins operate, and thus the resultant difficulties in estimating their energies and free energies. It is required to perform extensive sampling, properly treat the electronic structure of the bound metal or metals, and seamlessly merge the required techniques to assess energies and entropies, or their changes, for the entire system. Additionally, the machinery needs to be computationally affordable. Although a great advancement has been made over the years, including some of the seminal works resulting in the 2013 Nobel Prize in chemistry, many aforementioned exciting applications remain far from reach. We review the methodology on the forefront of the field, including several promising methods developed in our lab that bring us closer to the desired modern goals. We further highlight their performance by a few examples of applications

Inhibition Mechanism of SARS-CoV-2 Main Protease with Ketone-Based Inhibitors Unveiled by Multiscale Simulations

We present the results of combined classical and QM/MM simulations for the inhibition of SARS-CoV-2 3CL protease by a recently proposed ketone-based covalent inhibitor, PF-00835231, that is under clinical trial. In the noncovalent complex formed after binding into the active site the carbonyl group of this inhibitor is accommodated into the oxyanion hole formed by the NH main chain groups of residues 143 to 145. The P1-P3 groups of the inhibitor establish similar interaction with the enzyme to those of equivalent groups in the natural peptide substrate, while the hydroxymethyl moiety of the inhibitor partly mimics the interactions established by the P1’ group of the peptide in the active site. Regarding the formation of the covalent complex, the reaction is initiated after the proton transfer from Cys145 to His41. Formation of the covalent hemithioacetal complex takes place by means of the nucleophilic attack of the Sg atom of Cys145 on the electron deficient carbonyl carbon atom and a proton transfer from the catalytic His41 to the carbonyl oxygen atom mediated by the hydroxyl group. Our findings can be used as a guide to propose modifications of the inhibitor in order to increase its affinity by the 3CL protease.</p

Multiscale Simulations of SARS-CoV-2 3CL Protease Inhibition with Aldehyde Derivatives. Role of Protein and Inhibitor Conformational Dynamics in the Reaction Mechanism

We here investigate the mechanism of SARS-CoV-2 3CL protease inhibition by one of the most promising families of inhibitors, those containing an aldehyde group as warhead. These compounds are covalent inhibitors that inactivate the protease forming a stable hemithioacetal complex. Inhibitor 11a is a potent inhibitor that has been already tested in vitro and in animals. Using a combination of classical and QM/MM simulations we determined the binding mode of the inhibitor into the active site and the preferred rotameric state of the catalytic histidine. In the noncovalent complex the aldehyde group is accommodated into the oxyanion hole formed by the NH main chain groups of residues 143 to 145. In this pose, P1-P3 groups of the inhibitor mimic the interactions established by the natural peptide substrate. The reaction is initiated with the formation of the catalytic dyad ion pair after a proton transfer from Cys145 to His41. From this activated state, covalent inhibition proceeds with the nucleophilic attack of the deprotonated Sg atom of Cys145 to the aldehyde carbon atom and a water mediated proton transfer from the Ne atom of His41 to the aldehyde oxygen atom. Our proposed reaction transition state structure is validated by comparison with x-ray data of recently reported inhibitors, while the activation free energy obtained from our simulations agrees with the experimentally derived value, supporting the validity of our findings. Our study stresses the interplay between the conformational dynamics of the inhibitor and the protein with the inhibition mechanism and the importance of including conformational diversity for accurate predictions about the inhibition of the main protease of SARS-CoV-2. The conclusions derived from our work can also be used to rationalize the behavior of other recently proposed inhibitor compounds, including aldehydes and ketones with high inhibitory potency.</p