ENZYME EFFECTOR DESIGN

酶效应器设计

• 批准号: 3269943
• 负责人: RICHARD L. SCHOWEN
• 金额: $ 12.6万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3269943
• 关键词: Bacillus; Pseudomonas; carbon; chemical kinetics; chemical structure function; chymotrypsin; deuterium; enzyme activity; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; lactate dehydrogenases; nonradiation isotope effect; oxygen; peptidases; pyruvate decarboxylase; radiotracer; stable isotope; thermodynamics

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.

该假设将被检验，酶的催化能力 起源于生物之间共同进化的相互作用 特定于酶衍生和底物衍生的结构 催化过渡态相互作用要么不存在 或在反应物和中间状态下减弱。 这些动作 应该是“进化预期”最强的 酶/底物组合并在一定程度上被禁用 “非天然”底物或突变酶。 三种酶 将学习的课程：丝氨酸蛋白酶、丙酮酸 脱羧酶和乳酸脱氢酶。 质子库存 随着肽链长度增加的底物将 决定测试 Asp-i02-Asn 的多质子催化作用 胰蛋白酶的突变体，这将揭示多质子是否 催化作用由或不由电荷中继系统产生。 根据结果​​，其他突变胰蛋白酶也可能是 研究过。 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的作用将是 测量以推断“物理”步骤和 “化学”步骤确定速率，并探测 量子隧道效应在酶酸碱催化中的重要性。 用丙酮酸脱羧酶捕获碳负离子 中间体将用于确定内在同位素效应 用于脱羧步骤和准确的自由能图 将被建造。 与酶的相互作用 将研究类底物抑制剂 HCOCOOH 和 CO(COOH)2 动力学和溶剂同位素效应。 乳酸 来自嗜冷、中温和嗜热的脱氢酶 将通过初级和次级同位素效应来研究细菌 确定氨基酸序列如何发生变化的方法 热适应反映在过渡态 结构和各个步骤的相关大鼠。 溶剂 同位素效应将探讨亲电子的重要性 氢化物转移和底物同位素的催化作用 隧道掘进的作用。 嗜热酶的突变体也会 被检查。 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器。