Modeling the Catalysis of Anti-Cocaine Catalytic Antibody: Competing Reaction Pathways and Free Energy Barriers

Abstract

The competing reaction pathways and the corresponding free energy barriers for cocaine hydrolysis catalyzed by an anti-cocaine catalytic antibody, mAb15A10, were studied by using a novel computational strategy based on the binding free energy calculations on the antibody binding with cocaine and transition states. The calculated binding free energies were used to evaluate the free energy barrier shift from the cocaine hydrolysis in water to the antibody-catalyzed cocaine hydrolysis for each reaction pathway. The free energy barriers for the antibody-catalyzed cocaine hydrolysis were predicted to be the corresponding free energy barriers for the cocaine hydrolysis in water plus the calculated free energy barrier shifts. The calculated free energy barrier shift of -6.87 kcal/mol from the dominant reaction pathway of the cocaine benzoyl ester hydrolysis in water to the dominant reaction pathway of the antibody-catalyzed cocaine hydrolysis is in good agreement with the experimentally derived free energy barrier shift of -5.93 kcal/mol. The calculated mutation-caused shifts of the free energy barrier are also reasonably close to the available experimental activity data. The good agreement suggests that the protocol for calculating the free energy barrier shift from the cocaine hydrolysis in water to the antibody-catalyzed cocaine hydrolysis may be used in future rational design of possible high-activity mutants of the antibody as anti-cocaine therapeutics. The general strategy of the free energy barrier shift calculation may also be valuable in studying a variety of chemical reactions catalyzed by other antibodies or proteins through noncovalent bonding interactions with the substrates.

Design, Preparation, and Characterization of High-Activity Mutants of Human Butyrylcholinesterase Specific for Detoxification of Cocaine

“…Finally, the measured (Ϫ)-cocaine concentration-dependent radiometric data were analyzed by using the standard Michaelis-Menten kinetics so that the catalytic parameters were determined along with the use of a well established standard enzyme-linked immunosorbent assay protocol (Pan et al, 2008 …”

“…Therefore, starting from the A328W/Y332A or A328W/ Y332G mutant, rational design of BChE mutants against (Ϫ)-cocaine has been focused on decreasing the energy barrier for the first reaction step without significantly affecting the other reaction steps. We have developed unique computational strategies and protocols based on the virtual screening of rate-determining transition states of the enzymatic reaction to design enzyme mutants with improved catalytic activity (Pan et al, 2005(Pan et al, , 2007(Pan et al, , 2008Zheng et al, 2008Zheng et al, , 2010Yang et al, 2009). The computational design was followed by in vitro experiments, including site-directed mutagenesis, protein expression, and fast enzyme activity screening using the culture medium.…”

“…The computational design was followed by in vitro experiments, including site-directed mutagenesis, protein expression, and fast enzyme activity screening using the culture medium. The integrated computational-experimental studies have led to the discovery of some BChE mutants with a significantly improved catalytic efficiency against (Ϫ)-cocaine (Pan et al, 2005(Pan et al, , 2007(Pan et al, , 2008Zheng et al, 2008;Yang et al, 2009;Yang et al, 2010). One of our designed and discovered high-activity mutants of human BChE (i.e., the A199S/S287G/A328W/Y332G mutant) (Pan et al, 2005) has been validated by an independent group of scientists who concluded that this mutant is "a true cocaine hydrolase with a catalytic efficiency that is 1000-fold greater than wild-type BChE.…”

Desialylation of dying cells with catalytically active antibodies possessing sialidase activity facilitate their clearance by human macrophages

“…DANA is a sialidase inhibitor, an uncleavable analogue of transitional state intermediate during sialic acid trimming [27]. Recent studies have shown that by utilizing haptens for immunization one can induce abzymes with exotic specificities, such as hydrolyzing cocaine [28,29] or organophosphorus nerve poisons [30].…”

Long-acting cocaine hydrolase for addiction therapy

“…It has been difficult to improve the CAb activity further because a CAb, unlike an enzyme, can help stabilize the transition state only for nonenzymatic cocaine hydrolysis; a mechanistic study (20) indicates there is no formation of a covalent bond or breaking between the substrate and CAb during the reaction process.…”