KINETIC STUDIES OF ENZYME MECHANISMS

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。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。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，低势垒的氢键来获得催化所需的能量，而在基态只有弱氢键。低势垒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这样的低场信号，我们认为，从其他证据来看，它们应该存在。例如，我们将研究与氯富马酸盐、3-硝基乳酸盐或3-硝基丙酸盐复合的脱氢酶，因为这种复合物应该模拟反应中烯醇化的“碳负离子”中间体，我们认为它必须通过低势垒氢键来稳定。低势垒氢键也具有低氘分馏因子，其可以通过在混合的H2O和D2 O溶剂中对NMR峰进行积分来测量。我们将测量在天冬氨酸酶复合物中发现的任何低场峰的分馏因子，以及先前在天冬氨酸转氨酶的NMR光谱中看到的低场峰的分馏因子。

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.