KINETIC STUDIES OF ENZYME MECHANISMS

酶机制的动力学研究

• 批准号: 7420538
• 负责人: WILLIAM Wallace CLELAND
• 金额: $ 0.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-20 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7420538
• 关键词: enzymes; hydro lyase; hydrogen bond

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. The purpose of this project is to seek experimental evidence to support the recent hypothesis that enzymes obtain the energy needed for catalysis by forming very strong, low-barrier, hydrogen bonds in the transition state, or in otherwise unstable intermediates, while having only weak hydrogen bonds in the ground state. Low-barrier hydrogen bonds have very low field proton NMR chemical shifts, and we will look for such low field signals in enzyme complexes where we think, from other evidence, that they should be present. For example, we will look at fumarase complexed with chlorofumarate, 3-nitrolactate, or 3-nitropropionate, since such complexes should mimic the enolized "carbanion" intermediate in the reaction which we believe must be stabilized by low-barrier hydrogen bonds. Low-barrier hydrogen bonds also have low deuterium fractionation factors, which can be measured by integrating the NMR peaks in mixed H2O and D2O solvent. We will measure the fractionatio n factors of any low field peaks we find in fumarase complexes, and also the fractionation factors of the low field peaks previously seen in the NMR spectra of aspartate transaminase.

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。该项目的目的是寻找实验证据来支持最近的假设，即酶通过在过渡态或其他不稳定的中间体中形成非常强的、低势垒的氢键，同时仅具有弱氢来获得催化所需的能量基态键。低势垒氢键具有非常低的场质子核磁共振化学位移，我们将在酶复合物中寻找如此低场的信号，从其他证据来看，我们认为它们应该存在。例如，我们将研究与氯代富马酸盐、3-硝基乳酸或3-硝基丙酸盐复合的延胡索酶，因为此类复合物应模拟反应中的烯醇化“碳阴离子”中间体，我们认为该中间体必须通过低势垒氢键稳定。低势垒氢键还具有低氘分馏因子，可以通过在混合 H2O 和 D2O 溶剂中积分 NMR 峰来测量。我们将测量在延胡索酶复合物中发现的任何低场峰的分级因子，以及之前在天冬氨酸转氨酶的 NMR 谱中看到的低场峰的分级因子。