NOVEL COCAINE ESTERASES

本建议书中所述的工作是申请KO2的一部分。 研究科学家发展奖。成人丁酰胆碱酯酶 是一种关键的血清酶，可以水解和解毒许多异生物质 含有羧酸酯部分。虽然酶已经 研究了很多年，是该领域的起点， 令人惊讶的是，在药物遗传学中，酶的生理作用是 未知我们研究的长期目标是开发更有效的 用于水解和除去可卡因的解毒生物催化剂 人体组织这种反可卡因催化剂可以拯救生命。之甚少 已知人体丁酰胆碱酯酶氨基酸突变的影响 可卡因的水解作用丁酰胆碱酯酶是 保护人类免受潜在有毒化学物质的防御， 他们的环境。丁酰胆碱酯酶减少的人 活性可能倾向于异生物质的毒性化学作用 包括滥用药物。 拟议的研究分为四个主要部分： 用于动力学和机制研究的人丁酰胆碱酯酶变体， 2)丁酰胆碱酯酶作为解毒催化剂的评价 通过新的生物分析方法的可卡因水解，3）设计 丁酰胆碱酯酶变体与分子建模，和4）表达 中国人卵巢（CHO）细胞中新的活性变体。具体 第2节的目的包括验证重组体的活性， 酵素第2节的具体目标包括动力学评价 通过新的HPLC和放射性测定法测定变体重组酶。的 具体而言，第3节的目的包括研究丁酰胆碱酯酶， 使用分子建模和分子测量设备。具体目标 第4节中的突变体包括 CHO细胞中的丁酰胆碱酯酶。我们将检查 丁酰胆碱酯酶的催化活性。目前的结果 研究将提供一个详细的结构-功能的图片 人丁酰胆碱酯酶的关系。该研究将提供 了解丁酰胆碱酯酶在代谢中作用的基础 可卡因。这项工作的意义在于它将导致一个更 对可卡因中丁酰胆碱酯酶作用的深入了解 和其他滥用药物的解毒。

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.