TY - JOUR
T1 - Predicted Michaelis-Menten complexes of cocaine-butyrylcholinesterase
T2 - Engineering effective butyrylcholinesterase mutants for cocaine detoxication
AU - Sun, Hong
AU - El Yazal, Jamal
AU - Lockridge, Oksana
AU - Schopfer, Lawrence M.
AU - Brimijoin, Stephen
AU - Pang, Yuan Ping
PY - 2001/3/23
Y1 - 2001/3/23
N2 - Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser198 for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe 329 and Trp430. These interactions do not occur with (+)-cocaine. Because the rate of (-)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, 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. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.
AB - Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser198 for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe 329 and Trp430. These interactions do not occur with (+)-cocaine. Because the rate of (-)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, 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. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.
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U2 - 10.1074/jbc.M006676200
DO - 10.1074/jbc.M006676200
M3 - Article
C2 - 11104759
AN - SCOPUS:0035937713
SN - 0021-9258
VL - 276
SP - 9330
EP - 9336
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 12
ER -