ENZYME EFFECTOR DESIGN

这个假设将被验证，即酶的催化能力 起源于生物共同进化的相互作用， 酶衍生的和底物衍生的结构特异于 催化过渡态相互作用， 或在反应物和中间状态中弱化。 这些行动 应该是最强的“进化预期” 酶/底物组合，并在一定程度上与 “非天然”底物或突变酶。 三种酶 类将被研究：丝氨酸蛋白酶，丙酮酸 脱羧酶和乳酸脱氢酶。 质子存量 随着底物的肽链长度增加， 测定Asp-i 02-Asn的多质子催化试验 胰蛋白酶的突变体，这将揭示是否多质子 催化作用由或不由电荷中继系统产生。 根据结果，其他突变型胰蛋白酶也可能是 研究了 溶剂同位素的温度依赖性 对胰凝乳蛋白酶、胰蛋白酶、弹性蛋白酶和枯草杆菌蛋白酶影响将 测量以推断“物理”步骤和 “化学”步骤决定速率，并探测 量子隧穿在酶酸碱催化中重要性 用丙酮酸脱羧酶捕获负碳离子 中间体将用于确定固有同位素效应 以及精确的自由能图 将被建造。 与酶的相互作用 研究了HCOCOOH和CO（COOH）2两种底物类缓蚀剂 动力学和溶剂同位素效应。 乳酸 来自嗜冷、嗜温和嗜热的芽孢杆菌 细菌将通过初级和次级同位素效应进行研究 确定氨基酸序列如何发生变化的方法 反映在过渡态中 结构和个别步骤的相对大鼠。 溶剂 同位素效应将探测亲电的重要性 氢化物转移催化作用和底物同位素效应 隧道的作用。 嗜热酶的突变体也将 接受检查。 这些研究的结果最终应该是 可用于设计抑制或激活酶的效应物。

The hypothesis will be tested that the catalytic power of enzymes originates in biologically co-evolved interactions between enzyme-derived and substrate-derived structures specific to the catalytic transition state interactions which are either absent or weakened in reactant and intermediate states. These actions should be strongest with the "evolutionarily anticipated" enzyme/substrate combinations and disabled to some extent with "unnatural" substrates or mutant enzymes. Enzymes from three classes will be studied: serine proteases, pyruvate decarboxylase and lactate dehydrogenases. Proton inventories with substrates of increasing peptide chain length will be determined to test for multiproton catalysis by the Asp-i02-Asn mutant of trypsin, which will reveal whether the multiproton catalysis is or is not produced by the charge-relay system. Depending on the results, other mutant trypsins may also be studied. The temperature dependence of the solvent isotope effect for chymotrypsin, trypsin, elastase and subtilisin will be measured to deduce the degree to which "physical" steps and "chemical" steps determine the rate, and to probe for the importance of quantum tunneling in enzymic acid-base catalysis. With pyruvate decarboxylase, trapping of the carbanion intermediate will be used to determine intrinsic isotope effects for the decarboxylation step and an accurate free energy diagram will be constructed. The interaction with the enzyme of the substrate-like inhibitors HCOCOOH and CO(COOH)2 will be studied kinetically and by solvent isotope effects. Lactate dehydrogenases from psychrophilic, mesophilic and thermophilic bacteria will be studied by primary and secondary isotope-effect methods to determine how the amino-acid sequence changes arising from thermal adaptation are reflected in transitionstate structure and in the relative rats of individual steps. Solvent isotope effects will probe the importance of electrophilic catalysis of hydride transfer and substrate isotope effects the role of tunneling. A mutant of the thermophilic enzyme will also be examined. The results of these studies should eventually be useful in designing effectors to inhibit or activate enzymes.