Predicted Michaelis-Menten complexes of cocaine-butyrylcholinesterase - Engineering effective butyrylcholinesterase mutants for cocaine detoxication

Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hyd rolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-st ereoisomer, A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate stru ctural factors underlying the stereochemical difference in catalysis, Here, we report two three-dimensional Michaelis-Menten complexes of BChE ligande d with natural and unnatural cocaine molecules, respectively, that were der ived from molecular modeling and supported by experimental studies. Such co mplexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser(198) for hydrolysis, Rotation of (-)- cocaine appears to be hindered by interactions of its phenyl ring with Phe( 329) and Trp(430). These interactions do not occur with (+)-cocaine, Becaus e the rate of (-)-cocaine hydrolysis is predicted to be determined mainly b y the re-orientation step, it should not be greatly influenced by pH. In fa ct, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine, O ur models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocai ne, and they suggest that mutations of certain residues in the catalytic si te could greatly improve catalytic efficiency and the potential for detoxic ation.