Stereoselective inhibition of human butyrylcholinesterase by pbosphonotbiolate analogs of (+)- and (−)-cocaine

Berkman, Clifford E.; Underiner, Gail E.; Cashman, John R.

doi:10.1016/s0006-2952(97)00403-6

Cited by 16 publications

(19 citation statements)

References 19 publications

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“…Although cocaine's two isomers differ more than 1000-fold in rates of hydrolysis, wild-type BChE binds them nearly equally well (Gatley, 1991;Berkman et al, 1997;Xie et al, 1999;Sun et al, 2001). We argue that BChE's low efficiency with (Ϫ)-cocaine is caused not by weak binding or poor catalysis per se but by difficulty in moving substrate from binding site to active site.…”

Section: Discussionmentioning

confidence: 99%

Re-engineering Butyrylcholinesterase as a Cocaine Hydrolase

Sun

Pang

Lockridge

et al. 2002

Molecular Pharmacology

134

170

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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 ϭ 4 min Ϫ1 ) 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 ϭ 154 min Ϫ1

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Section: Discussionmentioning

confidence: 99%

Re-engineering Butyrylcholinesterase as a Cocaine Hydrolase

Sun

Pang

Lockridge

et al. 2002

Molecular Pharmacology

134

170

View full text Add to dashboard Cite

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“…Nevertheless, the literature provides no explanation of how cocaine forms its Michaelis-Menten complex with BChE or why BChE hydrolyzes the unnatural stereoisomer much faster than the natural cocaine although their K m values are similar (9,17,18). Addressing these questions with a computational approach should reduce the need to construct and evaluate many mutant enzymes in a random search for an effective cocaine hydrolase.…”

mentioning

confidence: 99%

Predicted Michaelis-Menten Complexes of Cocaine-Butyrylcholinesterase

Sun¹,

Yazal²,

Lockridge

et al. 2001

Journal of Biological Chemistry

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Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (؊)-cocaine only one-two thousandth as fast as the unnatural (؉)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser 198 for hydrolysis. Rotation of (؊)-cocaine appears to be hindered by interactions of its phenyl ring with Phe 329 and Trp 430 . These interactions do not occur with (؉)-cocaine. Because the rate of (؊)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (؉)-cocaine. Our models can explain why BChE hydrolyzes (؉)-cocaine faster than (؊)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.Cocaine overdose is a leading cause of death among urbandwelling young adults (1, 2). A new idea for treating cocaine overdose is to accelerate metabolic clearance by administering an appropriate hydrolase. Plasma butyrylcholinesterase (BChE) 1 can hydrolyze cocaine to ecgonine methyl ester and reportedly accounts for all the blood-borne cocaine hydrolysis activity in humans (3-5). Pretreatment with human BChE provides rats with substantial protection against cocaine-induced cardiac arrhythmia, hypertension, and locomotor hyperactivity (6). Exogenous BChE is also able to rescue rats given an overdose of cocaine (7). However, the k cat of BChE for (Ϫ)-cocaine is only 3.9 min Ϫ1, versus 33,900 min Ϫ1 for the optimal substrate, butyrylthiocholine. We estimate that timely detoxication of a cocaine overdose, with serum levels up to 20 mg/l (8), would require at least 100 mg of enzyme. An A328Y mutant of BChE developed by the trial and error approach (9) showed 4 -5-fold improvement in (Ϫ)-cocaine hydrolysis, but it is still not satisfactory for treatment of cocaine overdose. Because human BChE hydrolyzes synthetic (ϩ)-cocaine (see Fig. 1) about 2,000-fold faster than the natural (Ϫ)-cocaine (see Fig. 1) (9), elucidation of the structural factors responsible for the different catalytic rates may offer a rational strategy for engineering BChE mutants that can hydrolyze (Ϫ)-cocaine rapidly enough to treat cocaine overdose.Theoretical 3D structures of BChE (10 -12), of its complex with cocaine (9), and of the cocaine molecule itself have all been reported (13-16). Nevertheless, the literature provides no explanation o...

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“…BuChE detoxifies them before they reach the AChE at physiologically important target sites (Doctor and Saxena, 2005). BuChE plays an important role in the cocaine metabolism (Xie et al, 1999;Berkman et al, 1997;Mattes et al, 1997). Both ChEs are sensitive to carbamates and organophosphates, which are used as insecticides, nemacitides and chemical warfare agents.…”

Section: Introductionmentioning

confidence: 99%