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QM/MM Description of Newly Selected Catalytic Bioscavengers Against Organophosphorus Compounds Revealed Reactivation Stimulus Mediated by Histidine Residue in the Acyl-Binding Loop.

ABSTRACT: Butyrylcholinesterase (BChE) is considered as an efficient stoichiometric antidote against organophosphorus (OP) poisons. Recently we utilized combination of calculations and ultrahigh-throughput screening (uHTS) to select BChE variants capable of catalytic destruction of OP pesticide paraoxon. The purpose of this study was to elucidate the molecular mechanism underlying enzymatic hydrolysis of paraoxon by BChE variants using hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. Detailed analysis of accomplished QM/MM runs revealed that histidine residues introduced into the acyl-binding loop are always located in close proximity with aspartate residue at position 70. Histidine residue acts as general base thus leading to attacking water molecule activation and subsequent SN2 inline hydrolysis resulting in BChE reactivation. This combination resembles canonical catalytic triad found in active centers of various proteases. Carboxyl group activates histidine residue by altering its pKa, which in turn promotes the activation of water molecule in terms of its nucleophilicity. Observed re-protonation of catalytic serine residue at position 198 from histidine residue at position 438 recovers initial configuration of the enzyme's active center, facilitating next catalytic cycle. We therefore suggest that utilization of uHTS platform in combination with deciphering of molecular mechanisms by QM/MM calculations may significantly improve our knowledge of enzyme function, propose new strategies for enzyme design and open new horizons in generation of catalytic bioscavengers against OP poisons.

SUBMITTER: Zlobin A

PROVIDER: S-EPMC6085465 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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QM/MM Description of Newly Selected Catalytic Bioscavengers Against Organophosphorus Compounds Revealed Reactivation Stimulus Mediated by Histidine Residue in the Acyl-Binding Loop.

Zlobin Alexander A Mokrushina Yuliana Y Terekhov Stanislav S Zalevsky Arthur A Bobik Tatiana T Stepanova Anastasiya A Aliseychik Maria M Kartseva Olga O Panteleev Sergey S Golovin Andrey A Belogurov Alexey A Gabibov Alexander A Smirnov Ivan I

Frontiers in pharmacology 20180803

Butyrylcholinesterase (BChE) is considered as an efficient stoichiometric antidote against organophosphorus (OP) poisons. Recently we utilized combination of calculations and ultrahigh-throughput screening (uHTS) to select BChE variants capable of catalytic destruction of OP pesticide paraoxon. The purpose of this study was to elucidate the molecular mechanism underlying enzymatic hydrolysis of paraoxon by BChE variants using hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. D ...[more]

PMID: 30123127

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Project description:Organophosphorus agents (OPs) are irreversible inhibitors of acetylcholinesterase (AChE). OP poisoning causes major cholinergic syndrome. Current medical counter-measures mitigate the acute effects but have limited action against OP-induced brain damage. Bioscavengers are appealing alternative therapeutic approach because they neutralize OPs in bloodstream before they reach physiological targets. First generation bioscavengers are stoichiometric bioscavengers. However, stoichiometric neutralization requires administration of huge doses of enzyme. Second generation bioscavengers are catalytic bioscavengers capable of detoxifying OPs with a turnover. High bimolecular rate constants (kcat/Km > 106 M-1min-1) are required, so that low enzyme doses can be administered. Cholinesterases (ChE) are attractive candidates because OPs are hemi-substrates. Moderate OP hydrolase (OPase) activity has been observed for certain natural ChEs and for G117H-based human BChE mutants made by site-directed mutagenesis. However, before mutated ChEs can become operational catalytic bioscavengers their dephosphylation rate constant must be increased by several orders of magnitude. New strategies for converting ChEs into fast OPase are based either on combinational approaches or on computer redesign of enzyme. The keystone for rational conversion of ChEs into OPases is to understand the reaction mechanisms with OPs. In the present work we propose that efficient OP hydrolysis can be achieved by re-designing the configuration of enzyme active center residues and by creating specific routes for attack of water molecules and proton transfer. Four directions for nucleophilic attack of water on phosphorus atom were defined. Changes must lead to a novel enzyme, wherein OP hydrolysis wins over competing aging reactions. Kinetic, crystallographic, and computational data have been accumulated that describe mechanisms of reactions involving ChEs. From these studies, it appears that introducing new groups that create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states (TS), and intermediates may determine whether dephosphylation is favored over aging. Mutations on key residues (L286, F329, F398) were considered. QM/MM calculations suggest that mutation L286H combined to other mutations favors water attack from apical position. However, the aging reaction is competing. Axial direction of water attack is not favorable to aging. QM/MM calculation shows that F329H+F398H-based multiple mutants display favorable energy barrier for fast reactivation without aging.

Project description:In the current work, we found aqueous enzyme phase reaction pathways for the reactivation of the exogenously inhibited [Fe-Fe]-hydrogenases by O2, or OH(-), which metabolizes to H2O(1,2). We used the hybrid quantum mechanics/molecular mechanics (QM/MM) method to study the reactivation pathways of the exogenously inhibited enzyme matrix. The ONIOM calculations performed on the enzyme agree with experimental results(3), i.e., wild-type [Fe-Fe]-hydrogenase H-cluster is inhibited by oxygen metabolites. An enzyme spherical region with a radius of 8 Å (from the distal iron, Fed) has been screened for residues that prevent H2O from leaving the catalytic site and reactivate the [Fe-Fe]-hydrogenase H-cluster. In the screening process, polar residues were removed, one at a time, and frequency calculations provided the change in the Gibbs' energy for the dissociation of water (due to their deletion). When residue deletion resulted in significant Gibbs' energy decrease, further residue substitutions have been carried out. Following each substitution, geometry optimization and frequency calculations have been performed to assess the change in the Gibbs' energy for the elimination H2O. Favorable thermodynamic results have been obtained for both single residue removal (?G?Glu(374) = -1.6 kcal/mol), single substitution (?GGlu(374)His = -3.1 kcal/mol), and combined residue substitutions (?GArg(111)Glu;Thr(145)Val;Glu(374)His;Tyr(375)Phe = -7.5 kcal/mol). Because the wild-type enzyme has only an endergonic step to overcome, i.e., for H2O removal, by eliminating several residues, one at a time, the endergonic step was made to proceed spontaneously. Thus, the most promising residue deletions which enhance H2O elimination are ?Arg(111), ?Thr(145), ?Ser(177), ?Glu(240), ?Glu(374), and ?Tyr(375). The thermodynamics and electronic structure analyses show that the bridging carbonyl (COb) of the H-cluster plays a concomitant role in the enzyme inhibition/reactivation. In gas phase, COb shifts towards Fed to compensate for the electron density donated to oxygen upon the elimination of H2O. However, this is not possible in the wild-type enzyme because the protein matrix hinders the displacement of COb towards Fed, which leads to enzyme inhibition. However, enzyme reactivation can be achieved by means of appropriate amino acid substitutions.

Project description:A combined quantum mechanical/molecular mechanical (QM/MM) potential function is used in a thermodynamic integration approach to calculate the pK(a) of residue 66 in two mutants (V66E, V66D) of Staphylococal nuclease relative to solution. Despite the similarity in chemical nature and experimentally measured pK(a) of the two buried titritable residues, the behaviors of the two mutants and the computed pK(a) values vary greatly in the simulations. For Glu66, the side chain is consistently observed to spontaneously flip out from the protein interior during titration, and the overall protein structure remains stable throughout the simulations. The computed pK(a) shifts using conventional sampling techniques with multiple nanoseconds per lambda window (Set A and B) are generally close to the experimental value, therefore indicating that large-scale conformational rearrangements are not as important for V66E as suggested by the recent study of Warshel and co-worker. For Asp66, by contrast, flipping of the shorter side chain is not sufficient for getting adequate solvent stabilization of the ionized state. As a result, more complex behaviors such as partial unfolding of a nearby beta-sheet region is observed, and the computed pK(a) shift is substantially higher than the experimental value unless Asp66 is biased to adopt the similar configurations as Glu66 in the V66E simulations. Collectively, these studies suggest that the lack of electronic polarization is not expected to be the dominant source of error in microscopic pK(a) shift calculations, while the need of enhanced sampling is more compelling for predicting the pK(a) of buried residues. Furthermore, the comparison between V66E and V66D also highlights that the microscopic interpretation of similar apparent pK(a) values and effective "dielectric constants" of proteins can vary greatly in terms of the residues that make key contributions and the scale of structural/hydration response to titration, the latter of which is difficult to predict a priori. Perturbative analyses of interactions that contribute to the titration free energy point to mutants that can be used to verify the microscopic mechanisms of titration in V66E/D SNase proteins.

Dataset Information

QM/MM Description of Newly Selected Catalytic Bioscavengers Against Organophosphorus Compounds Revealed Reactivation Stimulus Mediated by Histidine Residue in the Acyl-Binding Loop.

Publications

QM/MM Description of Newly Selected Catalytic Bioscavengers Against Organophosphorus Compounds Revealed Reactivation Stimulus Mediated by Histidine Residue in the Acyl-Binding Loop.

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