TY - JOUR
T1 - Re-engineering butyrylcholinesterase as a cocaine hydrolase
AU - Sun, Hong
AU - Pang, Yuan Ping
AU - Lockridge, Oksana
AU - Brimijoin, Stephen
PY - 2002
Y1 - 2002
N2 - To address the problem of acute cocaine overdose, we undertook molecular engineering of butyrylcholinesterase (BChE) as a cocaine hydrolase so that modest doses could be used to accelerate metabolic clearance of this drug. Molecular modeling of BChE complexed with cocaine suggested that the inefficient hydrolysis (kcat = 4 min-1) involves a rotation toward the catalytic triad, hindered by Tyr332. To eliminate rotational hindrance and retain substrate affinity, we introduced two amino acid substitutions (Ala328Trp/Tyr332Ala). The resulting mutant BChE reduced cocaine burden in tissues, accelerated plasma clearance by 20-fold, and prevented cocaine-induced hyperactivity in mice. The enzyme's kinetic properties (kcat = 154 min-1, KM = 18 μM) satisfy criteria suggested previously for treating cocaine overdose (kcat > 120 min-1, KM < 30 μM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.
AB - To address the problem of acute cocaine overdose, we undertook molecular engineering of butyrylcholinesterase (BChE) as a cocaine hydrolase so that modest doses could be used to accelerate metabolic clearance of this drug. Molecular modeling of BChE complexed with cocaine suggested that the inefficient hydrolysis (kcat = 4 min-1) involves a rotation toward the catalytic triad, hindered by Tyr332. To eliminate rotational hindrance and retain substrate affinity, we introduced two amino acid substitutions (Ala328Trp/Tyr332Ala). The resulting mutant BChE reduced cocaine burden in tissues, accelerated plasma clearance by 20-fold, and prevented cocaine-induced hyperactivity in mice. The enzyme's kinetic properties (kcat = 154 min-1, KM = 18 μM) satisfy criteria suggested previously for treating cocaine overdose (kcat > 120 min-1, KM < 30 μM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.
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U2 - 10.1124/mol.62.2.220
DO - 10.1124/mol.62.2.220
M3 - Article
C2 - 12130672
AN - SCOPUS:0036077314
SN - 0026-895X
VL - 62
SP - 220
EP - 224
JO - Molecular pharmacology
JF - Molecular pharmacology
IS - 2
ER -