KINETIC ANALYSES OF SITE-SPECIFIC MUTANTS OF CARBOXYLESTERASES

羧基酯酶位点特异性突变体的动力学分析

• 批准号: 7381820
• 负责人: MATTHEW K ROSS
• 金额: $ 2.14万
• 依托单位: MISSISSIPPI STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381820
• 关键词: KINETIC; ANALYSES; SITE; SPECIFIC; MUTANTS

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。羧酸酯酶（CES）是混杂的代谢酶，可催化酯化的异种生物和内生元的水解。拟除虫菊酯杀虫剂是在美国东南部广泛发现的含有酯的环境污染物，因为它们在农业中广泛使用。当人类暴露于拟除虫菊酯时，CES是负责从体内排毒和清除这些化学物质的肝脏中的主要酶。但是，CES识别拟除虫菊酯并催化其水解的机制尚未完全表征。例如，苄氯菊酯是一种广泛使用的拟除虫菊酯，用作顺式和反异构体的混合物。这些异构体是通过CE酶立体选择性代谢的，例如，反异构体比顺式异构体裂解的速度明显快。 哪些选择性说明了这种选择性？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（称为PNB CE）中发现的羧酸酯酶具有81％的氨基酸序列身份，并且具有具有哺乳动物CE的高度结构同源性，兔肝羧酸酯酶（Wierdl等，2004）。重要的是，PNB CE不需要糖基化即可具有酶活性，并且可以在大肠杆菌中快速，方便地进行大量该酶的表达。因此，可以快速构建PNB CE cDNA中的序列变体，从而在与底物识别和催化有关的关键位置进行位点特异性氨基酸修饰酶。该试验项目的目的是使用野生型和PNB CE突变体研究拟除虫菊酯水解的催化机制。该研究的中心假设是：与野生型酶相比，突变的羧酸酯酶改变了底物结合亲和力和/或催化步骤，这些酶将提供对羧酸酯酶催化机制的见解。