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.
Highlights
De novo protein design based on methods of structural bioinformatics is an actively developing field of modern life science
All these findings clearly show the need for further development of design approaches to create new catalytic bioscavengers
The purpose of this study is to determine the molecular mechanism of paraoxonase activity mediated by newly described BChE variants using hybrid quantum mechanical/molecular mechanical (QM/molecular mechanics (MM)) calculations
Summary
De novo protein design based on methods of structural bioinformatics is an actively developing field of modern life science. Example of its practical application for the treatment of OP poisoning clearly demonstrated that OP binding was significantly decreased. This way the inhibition of endogenous BChE and AChE by OP will occur much earlier than interception of OP by bioscavenger, and no protective effect would be achieved (Malisi et al, 2009). That was demonstrated for mutant forms of paraoxonase and phosphotriesterase (Jbilo et al, 1994; Mesulam et al, 2002), which had a protective effect against 2 × LD50 cyclosarin (GF) and VX when the enzyme was administered at a dose of only 0.2 and 2 mg/kg, respectively
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