KINETIC ANALYSES OF SITE-SPECIFIC MUTANTS OF CARBOXYLESTERASES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。羧酸酯酶（CEs）是一种混杂代谢酶，可催化酯化的外源性和内源性生物的水解。拟除虫菊酯类杀虫剂是一种含酯的环境污染物，在美国东南部广泛发现，因为它们在农业工业中被广泛使用。当人类接触到拟除虫菊酯时，ce是肝脏中负责解毒和清除体内这些化学物质的主要酶。然而，CEs识别拟除虫菊酯并催化其水解的机理尚未完全确定。例如，氯菊酯是一种广泛使用的拟除虫菊酯，是顺式和反式异构体的混合物。这些异构体被CE酶立体选择性代谢，例如，反式异构体比顺式异构体裂解明显快。是什么导致了这种选择性？这是该试点项目正在解决的主要问题之一。最近，在枯草芽孢杆菌中发现的一种羧酸酯酶（称为pnb CE）被证明与哺乳动物CE兔肝羧酸酯酶具有81%的氨基酸序列一致性和高度的结构同源性（Wierdl et al., 2004）。重要的是，pnb CE不需要糖基化就具有酶活性，并且在大肠杆菌中可以快速方便地大量表达该酶。因此，可以快速构建pnb CE cDNA的序列变体，并在涉及底物识别和催化的关键位置进行特定位点的氨基酸修饰。本试验项目的目的是利用野生型和pnb CE突变体研究拟除虫菊酯类化合物水解的催化机制。本研究的中心假设是：与野生型酶相比，突变型羧酸酯酶改变了底物结合亲和力和/或催化步骤，这些酶将为深入了解羧酸酯酶的催化机制提供线索。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Carboxylesterases (CEs) are promiscuous metabolizing enzymes that catalyze the hydrolysis of esterified xenobiotics and endobiotics. The pyrethroid insecticides are ester-containing environmental pollutants widely found in the southeastern U.S. due to their extensive use in the agricultural industry. When humans are exposed to pyrethroids, CEs are the primary enzymes in the liver responsible for detoxifying and clearing these chemicals from the body. However, the mechanism by which CEs recognize pyrethroids and catalyze their hydrolysis is not completely characterized. For instance, permethrin is a widely used pyrethroid used as a mixture of cis- and trans-isomers. These isomers are stereoselectively metabolized by CE enzymes, for example the trans-isomer is cleaved markedly faster than the cis-isomer. What accounts for this selectivity? This is one of the primary questions being addressed by this pilot project. Recently, a carboxylesterase found in Bacillus subtilis (termed pnb CE) was shown to possess 81% amino acid sequence identity and a high degree of structural homology with a mammalian CE, rabbit liver carboxylesterase (Wierdl et al., 2004). Importantly, pnb CE did not need to be glycosylated to be enzymatically active and expression of large amounts of this enzyme can be done rapidly and conveniently in E. coli. Thus, sequence variants in pnb CE cDNA can be quickly constructed yielding enzymes with site-specific amino acid modifications at key locations involved in substrate recognition and catalysis. The objective of this pilot project will be to use wild type and pnb CE mutants to study the catalytic mechanism of pyrethroid hydrolysis. The central hypothesis for the research is: Mutant carboxylesterases have altered substrate binding affinities and/or catalytic steps compared with wild type enzyme, and these enzymes will provide insight into the catalytic mechanism of carboxylesterases.