KINETIC STUDIES OF ENZYME MECHANISMS

酶机制的动力学研究

• 批准号: 7598714
• 负责人: WILLIAM Wallace CLELAND
• 金额: $ 0.01万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598714
• 关键词: Acids; Agreement; Carbon; Computer Retrieval of Information on Scientific Projects Database; Equilibrium; Funding; Grant; Hydrogen; Hydrolysis; In Vitro; Institution; Isotopes; Kinetics; Left; Measures; Metals; Nitrogen; Object Attachment; Oxygen; Proteins; Purpose; Range; Rate; Research; Research Personnel; Resources; Solvents; Source; Time; United States National Institutes of Health; Water; base; disulfide bond; enzyme mechanism; fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether; formamide; protonation

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. Proteins can contain disulfide bonds or metal centers which can unfortunately oxidize over time when manipulated in vitro. The purpose of this project is to synthetize a compound that is a better oxidizer than those compounds currently available or an oxidizer that is more active in a pH range not now available. A compound must be both a good oxidizer and be soluble in water. Multiple isotope effects were measured at the reactive center of formamide during acid-catalyzed hydrolysis in water at 25 ¿C. The mechanism involves a rapid pre-equilibrium protonation of the carbonyl oxygen, followed by the formation of at least one symmetrical tetrahedral intermediate, which does not appreciably exchange its carbonyl oxygen with the solvent (kh/kex = 55). The pKa for formamide was determined by 15N NMR and found to be about -2.0. The formyl hydrogen kinetic isotope effect- is indicative of a transition state that is highly tetrahedral (Dkobs = 0.79); the carbonyl carbon kinetic isotope effect (13kobs = 1.031) is in agreement with this conclusion. The small leaving nitrogen kinetic isotope effect (15kobs = 1.0050) is consistent with some step prior to breaking the C-N bond as rate-determining. The carbonyl oxygen kinetic isotope effect (18kobs = 0.996) points to attack of water as the rate-determining step. On the basis of these results a mechanism is proposed in which attachment of the nucleophile to a protonated formamide molecule is rate-determining.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 蛋白质可能含有二硫键或金属中心，不幸的是，在体外操作时，它们会随着时间的推移而氧化，该项目的目的是合成一种比现有化合物更好的氧化剂或在 pH 范围内更具活性的氧化剂。现在还没有可用的化合物必须既是良好的氧化剂又可溶于水。 在 25 ¿ 水中酸催化水解过程中，在甲酰胺的反应中心测量了多种同位素效应C. 该机制涉及羰基氧的快速预平衡质子化，然后形成至少一种对称四面体中间体，该中间体不会明显地与溶剂交换其羰基氧（kh/kex = 55）。甲酰胺通过15N NMR测定并发现约为-2.0甲酰氢动力学同位素效应-表明过渡态是高度四面体的。 (Dkobs = 0.79)；羰基碳动力学同位素效应 (13kobs = 1.031) 与此结论一致，小的离开氮动力学同位素效应 (15kobs = 1.0050) 与断裂 C-N 键之前的某个步骤一致。确定羰基氧动力学同位素效应（18kobs = 0.996）指向水的攻击。根据这些结果，提出了一种机制，其中亲核试剂与质子化的甲酰胺分子的连接是速率决定的。