| Abstract
| - Molecular dynamics (MD) simulations were carried out to study cocaine binding with wild-type humanbutyrylcholinesterase (BChE) and its mutants based on a recently reported X-ray crystal structure of humanBChE. For each BChE−cocaine system, we simulated both the nonprereactive and prereactive complexes inwater. Despite the significant difference found at the acyl binding pocket, the simulated structures confirmthe fundamental structural and mechanistic insights obtained from earlier computational studies of wild-typeBChE with cocaine based on a homology model, e.g. the rate-determining step for BChE-catalyzed hydrolysisof biologically active (−)-cocaine is the (−)-cocaine rotation in the active site from the nonprereactive BChE−(−)-cocaine complex to the prereactive complex. It has been demonstrated that the MD simulations on boththe nonprereactive and prereactive BChE−cocaine complexes can clearly reveal whether specific mutationsproduce the desired BChE−(−)-cocaine binding structures in which the (−)-cocaine rotation is less hinderedwhile the required prereactive BChE−(−)-cocaine binding is maintained. Based on the MD simulations,both A328W/Y332A and A328W/Y332G BChE's are expected to have catalytic activity for (−)-cocainehydrolysis higher than that of wild-type BChE and the activity of A328W/Y332G BChE should be slightlyhigher than that of A328W/Y332A BChE due to the less-hindered (−)-cocaine rotation in the mutant BChE's.However, the less-hindered (−)-cocaine rotation is only a necessary condition for a higher activity mutantBChE. The (−)-cocaine rotation is also less hindered in A328W/Y332A/Y419S BChE, but (−)-cocaine bindswith A328W/Y332A/Y419S BChE in a way that is not suitable for the catalysis. Thus, A328W/Y332A/Y419S BChE is expected to lose the catalytic activity. The computational predictions were confirmed by ourexperimental kinetic data, demonstrating that the MD simulation-based computational protocol used in thisstudy is reliable in prediction of the catalytic activity of BChE mutants for (−)-cocaine hydrolysis.
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