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。该项目的目的是寻求实验证据，以支持最近的假设，即酶通过在过渡状态或其他不稳定的中间体中形成非常强，低键的氢键来获得催化所需的能量，而在地面状态下仅具有弱氢键。低级氢键具有非常低的场质子NMR化学位移，我们将在酶复合物中寻找如此低的田间信号，从其他证据来看，我们认为应该存在它们。例如，我们将研究与氯氟酸，3-亚硝酸酯或3-硝基原丙酸酯复合的富马酶，因为这样的复合物应模仿反应中的耐碳“ carbanion”中间体，我们认为必须通过低键氢键稳定。低级氢键也具有低氘分离因子，可以通过在混合H2O和D2O溶剂中整合NMR峰来衡量。我们将测量在富马酶复合物中发现的任何低场峰的分数因子，以及先前在天冬氨酸转氨酶NMR光谱中看到的低场峰的分馏因子。