Sun_2002_Mol.Pharmacol_62_220

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 microM) satisfy criteria suggested previously for treating cocaine overdose (k(cat) >120 min(-1), K(M) < 30 microM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.