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Automated docking and three-dimensional Quantitative Structure-Activity Relationship studies (3D QSAR) were performed for a series of 82 reversible, competitive and selective acetylcholinesterase (AChE) inhibitors. The suggested automated docking technique, making use of constraints taken from experimental crystallographic data, allowed to dock all the 82 substituted N-benzylpiperidines to the crystal structure of mouse AChE, because of short computational times. A 3D QSAR model was then established using the CoMFA method. In contrast to conventional CoMFA studies, the compounds were not fitted to a reference molecule but taken in their 'natural' alignment obtained by the docking study. The established and validated CoMFA model was then applied to another series of 29 N-benzylpiperidine derivatives whose AChE inhibitory activity data were measured under different experimental conditions. A good correlation between predicted and experimental activity data shows that the model can be extended to AChE inhibitory activity data measured on another acetylcholinesterase and/or at different incubation times and pH level.
Title: Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process Masson P, Fortier PL, Albaret C, Clery C, Guerra P, Lockridge O Ref: Chemico-Biological Interactions, 119-120:17, 1999 : PubMed
Wild-type (wt) butyrylcholinesterase (BuChE) and the E197D and D70G mutants were inhibited by diisopropylfluorophosphate (DFP) or soman under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process of DFP-phosphorylated enzymes (diisopropylphosphoryl-BuChE (DIP-BuChE)) was investigated. Hydrostatic pressure strongly increased the rate of aging of wt enzyme. The activation volumes (deltaV*) for the dealkylation reaction was -150 ml/mol for DIP-wtBuChE. On the other hand, pressure had little effect on the aging of the DIP-E197D mutant and no effect on the DIP-D70G mutant, indicating that the transition state of the aging reaction (dealkylation of an isoproxy chain) was associated with an extended conformation/hydration change in wtBuChE but not in mutants. The rate of aging decreased with osmotic pressure, supporting the idea that water is important for stabilizing the transition state. Molecular dynamics simulations were performed on the wtDIP adduct to relate the kinetic data to hydration changes in the enzyme active site gorge. The pH dependence of the melting temperature (Tm) of native and soman-wtBuChE, as determined by differential scanning calorimetry (DSC), indicated that the stabilization energy of aged BuChE is mainly due to the salt bridge between protonated H438 and PO-, with pK(H438) = 8.3. Electrophoresis under high pressure up to 2.5 kbar showed that aged wtBuChE did not undergo pressure-induced molten globule transition unlike the native enzyme. This transition was not seen for the mutant enzymes, indicating that mutants are resistant to the penetration of water into their structure. Our results support the conclusion that D70 and E197 are major residues for the water/H-bond network dynamics in the active site gorge of BuChE, both residues acting like valves. In mutant enzymes, mutated residues function like check valves: forced penetration of water in the gorge is difficult, release of water is facilitated.
Title: Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates Masson P, Xie W, Froment MT, Levitsky V, Fortier PL, Albaret C, Lockridge O Ref: Biochimica & Biophysica Acta, 1433:281, 1999 : PubMed
Human butyrylcholinesterase displays substrate activation with positively charged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site (PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and activation control, the single mutants D70G/Y and Y332F/A/D and the double mutants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, BTC and succinyldithiocholine, and the neutral ester o-nitrophenyl butyrate was measured. In addition, inhibition of wild-type and mutant enzymes by tetramethylammonium was investigated, at low concentrations of BTC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for activation control. The effects of double mutations on D70 and Y332 were complex. Double-mutant cycle analysis provided evidence for interaction between these residues. The category of interaction (either synergistic, additive, partially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity reflects both the cross-talk between residues involved in the sequential formation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS induce a conformational change in the active site gorge of butyrylcholinesterase that can alter both substrate binding and enzyme acylation.
Title: Hydration change during the aging of phosphorylated human butyrylcholinesterase: importance of residues aspartate-70 and glutamate-197 in the water network as probed by hydrostatic and osmotic pressures Masson P, Clery C, Guerra P, Redslob A, Albaret C, Fortier PL Ref: Biochemical Journal, 343:361, 1999 : PubMed
Wild-type human butyrylcholinesterase (BCHE) and Glu-197-->Asp and Asp-70-->Gly mutants (E197D and D70G respectively) were inhibited by di-isopropyl phosphorofluoridate under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process (dealkylation of an isopropyl chain) of phosphorylated enzymes [di-isopropylated (DIP)-BCHE] was investigated. Hydrostatic pressure markedly increased the rate of aging of wild-type enzyme. The average activation volume (DeltaV( not equal)) for the dealkylation reaction was -170 ml/mol for DIP wild-type BCHE. On the other hand, hydrostatic pressure had little effect on the aging of the DIP mutants (DeltaV( not equal)=-2.6 ml/mol for E197D and -2 ml/mol for D70G), suggesting that the transition state of the aging process was associated with an extended hydration and conformational change in wild-type BCHE, but not in the mutants. The rate of aging of wild-type and mutant enzymes decreased with osmotic pressure, allowing very large positive osmotic activation volumes (DeltaV not equal osm) to be estimated, thus probing the participation of water in the aging process. Molecular dynamics simulations performed on the active-site gorge of the wild-type DIP adduct showed that the isopropyl chain involved in aging was highly solvated, supporting the idea that water is important for stabilizing the transition state of the dealkylation reaction. Wild-type BCHE was inhibited by soman (pinacolyl methylphosphonofluoridate). Electrophoresis performed under high pressure [up to 2.5 kbar (1 bar=10(5) Pa)] showed that the soman-aged enzyme did not pass through a pressure-induced, molten-globule transition, unlike the native wild-type enzyme. Likewise, this transition was not seen for the native E197D and D70G mutants, indicating that these mutants are resistant to the penetration of water into their structure. The stability energetics of native and soman-aged wild-type BCHE were determined by differential scanning calorimetry. The pH-dependence of the midpoint transition temperature of endotherms indicated that the high difference in stabilization energy between aged and native BCHE (DeltaDeltaG=23.7 kJ/mol at pH 8.0) is mainly due to the salt bridge between protonated His-438 and PO(-), with pK(His-438)=8.3. A molecular dynamics simulation on the MIP adduct showed that there is no water molecule around the ion pair. The 'hydrostatic versus osmotic pressure' approach probed the importance of water in aging, and also revealed that Asp-70 and Glu-197 are the major residues controlling both the dynamics and the structural organization of the water/hydrogen-bond network in the active-site gorge of BCHE. In wild-type BCHE both residues function like valves, whereas in the mutant enzymes the water network is slack, and residues Gly-70 and Asp-197 function like check valves, i.e. forced penetration of water into the gorge is not easily achieved, thereby facilitating the release of water.
Title: Mechanical Aspects of the Phosphotriesterase Activity of Human Butyrylcholinesterase G117H Mutant Albaret C, Masson P, Broomfield CA, Kaim LE, Fortier PL Ref: In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases, (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp.:399, 1998 : PubMed
Title: Poster: Mechanical Aspects of the Phosphotriesterase Activity of Human Butyrylcholinesterase G117H Mutant Albaret C, Masson P, Broomfield CA, Kaim LE, Fortier PL Ref: In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases, (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp.:434, 1998 : PubMed
Although aspirin (acetylsalicylic acid) is negatively charged, it is hydrolysed by butyrylcholinesterase (BCHE). Catalytic parameters were determined in 100 mM Tris buffer, pH 7.4, in the presence and absence of metal cations. The presence of Ca2+ or Mg2+ (<100 mM) in buffer did not change the Km, but accelerated the rate of hydrolysis of aspirin by wild-type or D70G mutant BCHE by 5-fold. Turnover numbers were of the order of 5000-12000 min-1 for the wild-type enzyme and the D70G and D70K enzymes in 100 mM Tris, pH 7.4, containing 50 mM CaCl2 at 25 degreesC; Km values were 6 mM for wild-type, 16 mM for D70G and 38 mM for D70K. People with 'atypical' BCHE have the D70G mutation. The apparent inhibition seen at high aspirin concentration was not due to inhibition by excess substrate but to spontaneous hydrolysis of aspirin, causing inhibition by salicylate. The wild-type and D70G enzymes were competitively inhibited by salicylic acid; the D70K enzyme showed a complex parabolic inhibition, suggesting multiple binding. The effect of salicylate was substrate-dependent, the D70K mutant being activated by salicylate with butyrylthiocholine as substrate. Km value for wild-type enzyme was lower than for D70 mutants, suggesting that residue 70 located at the rim of the active site gorge was not the major site for the initial encounter aspirin-BCHE complex. On the other hand, the virtual absence of affinity of the W82A mutant for aspirin indicated that W82 was the major residue involved in formation of the Michaelis complex. Molecular modelling of aspirin binding to BCHE indicated perpendicular interactions between the aromatic rings of W82 and aspirin. Kinetic study of BCHE-catalysed hydrolysis of different acetyl esters showed that the rate limiting step was acetylation. The bimolecular rate constants for hydrolysis of aspirin by wild-type, D70G and D70K enzymes were found to be close to 1x106 M-1 min-1. These results support the contention that the electrostatic steering due to the negative electrostatic field of the enzyme plays a role in substrate binding, but plays no role in the catalytic steps, i.e. in the enzyme acetylation.
Title: Structural Changes in the Active Site Gorge of Phosphylated Cholinesterase Accompanying the Aging Process Masson P, Clery C, Guerra P, Fortier PL, Albaret C, Lockridge O Ref: In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases, (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp.:419, 1998 : PubMed
Herein a molecular mechanic study of the interaction of a lethal chemical warfare agent, O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (also called VX), with Torpedo californica acetylcholinesterase (TcAChE) is discussed. This compound inhibits the enzyme by phosphonylating the active site serine. The chirality of the phosphorus atom induces an enantiomeric inhibitory effect resulting in an enhanced anticholinesterasic activity of the SP isomer (VXS) versus its RP counterpart (VXR). As formation of the enzyme-inhibitor Michaelis complex is known to be a crucial step in the inhibitory pathway, this complex was addressed by stochastic boundary molecular dynamics and quantum mechanical calculations. For this purpose two models of interaction were analyzed: in the first, the leaving group of VX was oriented toward the anionic subsite of TcAChE, in a similar way as it has been suggested for the natural substrate acetylcholine; in the second, it was oriented toward the gorge entrance, placing the active site serine in a suitable position for a backside attack on the phosphorus atom. This last model was consistent with experimental data related to the high inhibitory effect of this compound and the difference in activity observed for the two enantiomers.
Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.
