Re-engineering butyrylcholinesterase as a cocaine hydrolase.
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.
Sun
Hong
H
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic & Foundation for Medical Education and Research, Rochester, Minnesota 55905, USA.
Pang
Yuan-Ping
YP
Lockridge
Oksana
O
Brimijoin
Stephen
S
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D002091:Butyrylcholinesterase / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D003042:Cocaine / Q000378:metabolism*; D006224:Cricetinae; D005818:Genetic Engineering; D006867:Hydrolases / Q000378:metabolism*; D008958:Models, Molecular; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D014443:Tyrosine; D014364:Tryptophan; D006867:Hydrolases; D002091:Butyrylcholinesterase; D003042:Cocaine; D000409:Alanine