Author Report for: Varfolomeev SD

A kinetic model describing the dynamics of synaptic "discharge" taking into account the kinetics of the injection of the neurotransmitter into the synaptic cleft, the pH-dependence of the catalytic activity of the enzyme, and diffusion withdrawal of protons is proposed. The model provides a physicochemical explanation for a number of important physiological phenomena, such as the neuromuscular paralysis, the molecular mechanism of neurological memory, and the effect of some neurotoxins and drugs.

In the framework of the kinetic model, the functioning of the cholinergic synapse is considered. The results of mathematical modeling of changes in the level of acetylcholine, induced pH impulse, the influence of the frequency of impulse transmission and inhibition of acetylcholinesterase are presented. Physicochemical explanation for a number of important physiological phenomena, such as neuromuscular paralysis, the molecular mechanism of neurological memory, and actions of nerve poisons and toxins, is given.

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198.H438.E325) was replaced by S198.H438.N322E in silico. Molecular dynamics for 1.5 mus and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198.H438.N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

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 > 10(6) M(-1)min(-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.

Conformational dynamics of wild-type human butyrylcholinesterase (BChE), two mutants of residue Ala328, the catalytically active Ala328Cys, and the catalytically inactive (silent) Ala328Asp, and their interactions with butyrylcholine were studied. The aim was to understand the molecular mechanisms by which point mutations may lead to silent BChE variant or alter catalytic activity. Importance of BChE natural variants is due to medical consequences, i.e. prolonged apnea, following administration of the myorelaxant esters, succinylcholine and mivacurium. Comparison of molecular dynamics (MD) simulations for the three model systems showed that: 1) the active mutant Ala328Cys mutant has some changes in configuration of catalytic residues, which do not prevent binding of butyrylcholine to the active site; 2) in the naturally-occurring silent variant Ala328Asp, the Asp328 carboxylate may either form a salt bridge with Lys339 or a H-bond with His438. In the first case, the Omega-loop swings off the gorge, disrupting the pi-cation binding site and the catalytic triad. In the second case, binding of cationic substrates in the catalytic center is also impaired. MD simulations carried out in 0.15 M NaCl, close to physiological ionic strength conditions, favored the second situation. It was seen that Asp328 forms a H-bond with the catalytic triad His438, which in turn disrupts the catalytic machinery. Therefore, we concluded that the Ala328Asp variant is not catalytically active because of that dramatic event. Computational results, consistent with in vitro biochemical data and clinical observations, validate our MD approach.

Cholinesterases display a hysteretic behavior with certain substrates and irreversible inhibitors. For years, this behavior has remained puzzling. However, several lines of evidence indicated that it is caused by perturbation of the catalytic triad and its water environment. In the present study, using molecular dynamics simulations of Ala328Cys BCHE mutant and wild-type BCHE in the absence and presence of a co-solvent (sucrose, glycerol), we provide evidence that hysteresis originates in a flip of the catalytic triad histidine (His438). This event is controlled by water molecules that interact with active site residues. The physiological significance of this phenomenon is still an issue.

We re-visited the results of quantum mechanics - molecular mechanics (QM/MM) approaches aiming to construct the reaction energy profile for the acylation stage of acetylcholine hydrolysis by acetylcholinesterase. The main emphasis of this study was on the energy of the first tetrahedral intermediate (TI) relative to the level of the enzyme-substrate (ES) complex for which contradictory data from different works had been reported. A new series of stationary points on the potential energy surface was calculated by using electronically embedding QM/MM schemes when starting from the crystal structure mimicking features of the reaction intermediate (PDB ID: 2VJA). A thoughtful analysis allows us to conclude that the energy of TI should be lower than that of ES, and a proper treatment of contributions from the oxyanion hole residues accounts for their relative positions.

Research on cholinesterases and effects of their inhibition in the USSR and Russia since 1930-1940s till present is exposed in historical aspects. The first physiological and toxicological effects of cholinesterase inhibition were reported by Alexander Ginetsinsky during World War II, when academic institutions were evacuated from Leningrad to Kazan. The main scientific schools that initiated research on chemistry, enzymology and physiology of cholinesterases and their inhibitors were leaded by Alexandr and Boris Arbuzovs, Victor Rozengart, Viktor Yakovlev, Michael Michelson, Martin Kabachnik, Mikhail Voronkov, Ivan Knunyants, Alexandr Bretskin and others. They investigated the main physiological effects of cholinesterase inhibitors, and analyzed the catalytic mechanisms of cholinesterases and related enzymes. Their contributions are landmarks in the history of cholinesterase research. At the present time revival of research on cholinesterases in different universities and institutes is vivid, in particular at the Moscow State University, research institutes of Russian Academy of Sciences and Kazan Scientific Center.

The combined quantum mechanical-molecular mechanical (QM/MM) based computational scheme for modeling the structure-reaction rate correlations was elaborated for the hydrolysis of the set of neutral esters in the active site of acetylcholinesterase (AChE). The energy barriers of hydrolysis were estimated on the basis of the equilibrium geometry configurations of the enzyme-substrate (ES) complexes. The obtained correlation between the rate of hydrolysis and the hydrophobicity of the substrate leaving group is consistent with experimental data. The developed method can be used to predict the substrate reactivity and to interpret the specific nature of the enzyme catalysis.

The reaction mechanism of acetylcholine hydrolysis by acetylcholinesterase, including both acylation and deacylation stages from the enzyme-substrate (ES) to the enzyme-product (EP) molecular complexes, is examined by using an ab initio type quantum mechanical - molecular mechanical (QM/MM) approach. The density functional theory PBE0/aug-6-31+G* method for a fairly large quantum part trapped inside the native protein environment, and the AMBER force field parameters in the molecular mechanical part are employed in computations. All reaction steps, including the formation of the first tetrahedral intermediate (TI1), the acylenzyme (EA) complex, the second tetrahedral intermediate (TI2), and the EP complex, are modeled at the same theoretical level. In agreement with the experimental rate constants, the estimated activation energy barrier of the deacylation stage is slightly higher than that for the acylation phase. The critical role of the non-triad Glu202 amino acid residue in orienting lytic water molecule and in stabilizing the second tetrahedral intermediate at the deacylation stage of the enzymatic process is demonstrated.