Journal
BIOPHYSICAL JOURNAL
Volume 96, Issue 5, Pages 1931-1938Publisher
CELL PRESS
DOI: 10.1016/j.bpj.2008.11.051
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Funding
- NIDA NIH HHS [R01 DA013930] Funding Source: Medline
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It is recognized that an ideal anti-cocaine treatment is to accelerate cocaine metabolism by producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., butyrylcholinesterase (BChE)-catalyzed hydrolysis of cocaine. BChE mutants with a higher catalytic activity against (-)-cocaine are highly desired for use as an exogenous enzyme in humans. To develop a rational design for high-activity mutants, we carried out free-energy perturbation (FEP) simulations on various mutations of the transition-state structures in addition to the corresponding free-enzyme structures by using an extended FEP procedure. The FEP simulations on the mutations of both the free-enzyme and transition-state structures allowed us to calculate the mutation-caused shift of the free-energy change from the free enzyme (BChE) to the transition state, and thus to theoretically predict the mutation-caused shift of the catalytic efficiency (k(cat)/K-m). The computational predictions are supported by the kinetic data obtained from the wet experiments, demonstrating that the FEP-based computational design approach is promising for rational design of high-activity mutants of an enzyme. One of the BChE mutants designed and discovered in this study has an similar to 1800-fold improved catalytic efficiency against (-)-cocaine compared to wild-type BChE. The high-activity mutant may be therapeutically valuable.
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