This project focuses on the rational redesign of human butyrylcholinesterase (BChE) in order to accelerate cocaine metabolism in man. Enhancing cocaine metabolism by administration of BChE has been recognized as a possible treatment strategy for cocaine overdose and addiction. However, the catalytic activity of this plasma enzyme is three orders-of-magnitude lower against the naturally occurring (-)-cocaine than that against the relatively biologically inactive (+)-cocaine isomer. (+)-cocaine can be cleared from plasma in seconds prior to partitioning into the central nervous system. The long-term goal of this project is to guide the construction of a BChE mutant using site-directed mutagenesis by our collaborators so that the mutant can be used as an exogenous enzyme in human body with a catalytic activity for (-)-cocaine comparable to that of wild-type BChE for (+)-cocaine. For this purpose, state-of-the-art computational techniques will be used to uncover the mechanistic difference of the enzymatic reaction between (-)-cocaine and (+)-cocaine and to establish a rational basis for theoretical design of BChE mutants with an improved activity for (-)-cocaine.
The specific aims of the investigation involved in the present proposal include: 1. To determine structures and dynamics of the BChE binding with (-)-cocaine and (+)-cocaine, including the non-prereactive and prereactive BChE-substrate complexes. 2. To uncover fundamental reaction pathways for BChE-catalyzed hydrolysis of (-)-cocaine and (+)-cocaine at the benzoyl ester by performing reaction coordinate calculations on properly chosen model systems; 3. To investigate dynamics and changes of BChE binding with (-)-cocaine and (+)-cocaine during the enzymatic hydrolysis by carrying out MD simulations on the enzyme-reactant, transition state, intermediate and product complexes; 4. To evaluate free energy profiles and kinetics of the enzymatic hydrolysis by performing combined quantum mechanical and free energy perturbation calculations (QM-FE); 5. To design BChE mutants expected to have higher activity for (-)-cocaine based on the binding information obtained from Specific Aim 2 and the energetic information from Specific Aim 3. ? ?
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