Abstract
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.
- The American Society for Pharmacology and Experimental Therapeutics
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