Vol. 62, Issue 2, 220-224, August 2002

Re-engineering Butyrylcholinesterase as a Cocaine Hydrolase

Hong Sun, Yuan-Ping Pang, Oksana Lockridge, and Stephen Brimijoin

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic & Foundation for Medical Education and Research (H.S., Y.-P.P., S.B.), Molecular Neuroscience Program, Mayo Graduate School (H.S., Y.-P.P., S.B.), and Mayo Clinic Cancer Center (Y.-P.P.), Rochester, Minnesota; and Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska (O.L.)

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 min¹) 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 min¹, K_M = 18 µM) satisfy criteria suggested previously for treating cocaine overdose (k_cat >120 min¹, K_M < 30 µM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.