NOVEL COCAINE ESTERASES

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The work described in this proposal is part of a request for an KO2 Research Scientist Development Award. The adult human butyrylcholinesterase is a key serum enzyme that hydrolyzes and detoxicates many xenobiotics containing a carboxylic acid ester moiety. While the enzyme has been studied for many years and was the starting point for the field of pharmacogenetics, surprisingly, the physiological role of the enzyme is unknown. The longterm goal of our research is to develop more effective detoxication biocatalysts for the hydrolysis and removal of cocaine from human tissues. Such anti-cocaine catalysts could be life-saving. Little is known about the effect of human butyrylcholinesterase amino acid mutations on cocaine hydrolysis. Butyrylcholinesterase is one of the components of the defense that protects humans against potentially toxic chemicals in their environment. Humans with a decreased amount of butyrylcholinesterase activity may be predisposed to the toxic chemical action of xenobiotics including drugs of abuse. The proposed studies are divided into four major sections: l) procurement of human butyrylcholinesterase variants for kinetic and mechanism studies, 2) Evaluation of butyrylcholinesterase as a detoxication catalyst for cocaine hydrolysis by novel bioanalytical methods, 3) Design of butyrylcholinesterase variants with molecular modeling, and 4) Expression of new, active variants in Chinese Hamster Ovary (CHO) cells. The specific aims of section 2 include the verification of the activity of recombinant enzyme. The specific aims of section 2 include the kinetic evaluation of the variant recombinant enzymes by novel HPLC and radiometric assays. The specific, aims of section 3 include the study of butyrylcholinesterase using molecular modeling and molecular measuring devices. The specific aims of section 4 include the expression of site-directed mutants of butyrylcholinesterase in CHO cells. We will examine the mutagenized butyrylcholinesterases for catalytic activity. The results of the present study will provide a detailed picture of the structure-function relationships of human butyrylcholinesterase. The study will provide a basis for understanding a role of butyrylcholinesterase in the metabolism of cocaine. The significance of the work is that it will lead to a more sophisticated understanding of a role of butyrylcholinesterase in cocaine and other drugs of abuse detoxication.