RT Journal Article
SR Electronic
T1 Re-engineering Butyrylcholinesterase as a Cocaine Hydrolase
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 220
OP 224
DO 10.1124/mol.62.2.220
VO 62
IS 2
A1 Hong Sun
A1 Yuan-Ping Pang
A1 Oksana Lockridge
A1 Stephen Brimijoin
YR 2002
UL http://molpharm.aspetjournals.org/content/62/2/220.abstract
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 (k cat = 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 (k cat = 154 min−1, K M = 18 μM) satisfy criteria suggested previously for treating cocaine overdose (k cat >120 min−1,K M < 30 μM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.