LOW BARRIER HYDROGEN BONDS IN ENZYMIC CATALYSIS

酶催化中的低势垒氢键

基本信息

批准号：
2634755
负责人：
WILLIAM Wallace CLELAND
金额：
$ 14.88万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-01-01 至 1998-12-31
项目状态：
已结题

项目摘要

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 low deuterium fractionation factors. Thus the low field proton NMR signals of chymotrypsin and trypsin that have been assigned to the proton between aspartate and histidine in the catalytic triad will be examined by proton and deuterium NMR to determine the chemical shift, the isotope effect on the chemical shift, and (by integrating the signal in an H2O-D2O mixture) the fractionation factor. This will be done under conditions where the histidine is protonated (low pH, or in the presence of tetrahedral complexes with specific inhibitors). Low-barrier hydrogen bonds will be characterized in model compounds that resemble the asparate-histidine structure in serine proteases, such as hydrogen cis-urocanate and hydrogen 2-(4-imidazole)-2-methylpropionate, using NMR and X-ray methods. The effect of internal low-barrier hydrogen bonds on the chemical reactivity of these compounds will be determined. A number of other compounds that potentially could form internal low- barrier hydrogen bonds in appropriate solvents will also be studied. Proton NMR will be used to look for low-barrier hydrogen bonds that stabilize "carbanion" intermediates in the reactions catalyzed by enolase and fumarase, using alternate substrates or inhibitors where previous kinetic studies indicate that the intermediate may be present in appreciable concentration at equilibrium. The signals will be characterized as outlined above.

该项目的目的是寻求实验证据来支持最近的假设是酶获得催化所需的能量通过在转变过程中形成非常强的、低势垒的氢键状态，或在其他不稳定的中间体中，同时只有弱氢键处于基态。低势垒氢键具有很强的低场质子核磁共振化学位移和低氘分馏因素。因此，胰凝乳蛋白酶的低场质子 NMR 信号和胰蛋白酶被分配给天冬氨酸和之间的质子催化三联体中的组氨酸将通过质子和氘进行检查 NMR 测定化学位移、同位素对化学物质的影响位移，并且（通过将信号积分到 H2O-D2O 混合物中）分馏因子。这将在以下条件下完成：组氨酸被质子化（低pH值，或在四面体存在的情况下）与特定抑制剂的复合物）。低势垒氢键将在模型化合物中表征：类似于丝氨酸蛋白酶中的天冬氨酸-组氨酸结构，例如顺式尿刊酸氢和2-(4-咪唑)-2-甲基丙酸氢，使用NMR和X射线方法。内部低势垒氢的影响键对这些化合物的化学反应性的影响将被确定。许多其他化合物可能会形成内部低还将研究适当溶剂中的势垒氢键。质子核磁共振将用于寻找低势垒氢键稳定烯醇酶催化反应中的“碳阴离子”中间体和富马酸酶，使用以前的替代底物或抑制剂动力学研究表明该中间体可能存在于平衡时有明显的浓度。信号将是其特征如上所述。