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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。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。列出的机构是 对于中心，这是调查员的机构。 蛋白质可以含有二硫键或金属中心，不幸的是，在体外操纵时，蛋白质可以随着时间的流逝。该项目的目的是合成比当前可用的化合物更好的氧化剂或在pH范围内更活跃的氧化剂更好的化合物。化合物必须既是良好的氧化剂，又是在水中固体。 在酸催化的水解在25€的水中的水解过程中，在甲酰胺的反应中心测量了多种同位素效应。该机制涉及羰基氧的快速前平衡质子化，然后形成至少一个对称的四面体中间体形成一个对象中的中间体，这不会与其carbonyl ocygen carbonyl oxygen（kh）（kh）（kh）kh/kh kh换成carbonyl氧气（Kh）。通过15N NMR确定形式的PKA，发现约为-2.0。甲基水动力学同位素效应 - 指示高度四面体的过渡态（DKOBS = 0.79）；羰基碳动力学同位素效应（13KOBS = 1.031）与该结论一致。小留下氮动力学同位素效应（15KOBS = 1.0050）在打破c-n键作为速率方面之前的一步是一致的。羰基氧动力学同位素效应（18KOBS = 0.996）指向水作为速率确定的步骤的攻击。根据这些结果，提出了一种机制，即将亲核试剂的附着在质子化的形式分子上是速率确定的。