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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。羧酸酯酶 (CE) 是一种混杂的代谢酶，可催化酯化的异生素和内生素的水解。拟除虫菊酯杀虫剂是一种含酯的环境污染物，由于在农业中的广泛使用，在美国东南部广泛存在。当人类接触拟除虫菊酯时，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，CE 识别拟除虫菊酯并催化其水解的机制尚未完全表征。例如，氯菊酯是一种广泛使用的拟除虫菊酯，用作顺式和反式异构体的混合物。这些异构体通过 CE 酶进行立体选择性代谢，例如反式异构体的裂解速度明显快于顺式异构体。 是什么造成了这种选择性？这是该试点项目要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% 的氨基酸序列同一性和高度的结构同源性（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表达大量该酶。因此，可以快速构建 pnb CE cDNA 中的序列变体，产生在参与底物识别和催化的关键位置进行位点特异性氨基酸修饰的酶。该试点项目的目标是利用野生型和 pnb CE 突变体来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，这些酶将提供对羧酸酯酶催化机制的深入了解。