LOW BARRIER HYDROGEN BONDS IN ENZYMIC CATALYSIS

酶催化中的低势垒氢键

• 批准号: 2022922
• 负责人: WILLIAM Wallace CLELAND
• 金额: $ 14.31万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/2022922
• 关键词: X ray crystallography; carbanion; catalyst; chemical kinetics; chemical synthesis; chromatography; chymotrypsin; deuterium; deuterium oxide; dicarboxylate; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; hydro lyase; hydrogen bond; nuclear magnetic resonance spectroscopy; phosphopyruvate hydratase; propionates; trypsin; urocanate

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以支持 最近的假设是酶获得催化所需的能量 通过在过渡中形成非常强的低垒氢键 声明或其他不稳定的中间体，而只有弱 氢键处于基态。 低级氢氢键非常 低场质子NMR化学位移和低氘分馏 因素。 因此，胰凝乳蛋白酶和 已分配给天冬氨酸和质子的胰蛋白酶 催化三合会中的组氨酸将通过质子和氘进行检查 NMR确定化学移位，同位素对化学的影响 移动，（通过将信号整合到H2O-D2O混合物中） 分馏因子。 这将在条件下完成 组氨酸被质子化（低pH或四面体存在 具有特定抑制剂的复合物）。 在模型化合物中将表征低垒氢键 类似于丝氨酸蛋白酶中的远大抗酸酯结构，例如 2-（4-咪唑）-2-甲基丙酸的氢顺式尿酸盐和氢气， 使用NMR和X射线方法。 内部低性氢的影响 将确定这些化合物的化学反应性的键。 许多其他可能形成内部低 - 的化合物 还将研究适当的溶剂中的屏障氢键。 质子NMR将用于寻找低屏障氢键 稳定烯醇酶催化的反应中的“ carbanion”中间体 和富马酶，使用以前的替代底物或抑制剂 动力学研究表明，中间体可能存在于 平衡时可观的浓度。 信号将是 表征如上所述。