ENZYME EFFECTOR DESIGN

这一假设将得到检验，即酶的催化能力 起源于生物共同进化的相互作用 特异性的酶衍生和底物衍生结构 不存在的催化过渡态相互作用 或在反应物和中间态被削弱。这些操作 应该是最强的与“进化的预期” 酶/底物组合并在一定程度上被禁用 “非天然”底物或突变酶。来自三种酶的酶 课程包括：丝氨酸蛋白酶、丙酮酸 脱羧酶和乳酸脱氢酶。质子库存 随着底物多肽链长度的增加， 用Asp-I02-ASN测定多质子催化性能 胰酶的突变体，这将揭示多质子 电荷继电器系统产生或不产生催化作用。 根据结果，其他突变的胰酶也可能是 学习。溶剂同位素的温度依赖性 对凝乳酶、胰蛋白酶、弹力酶和枯草杆菌酶的影响将是 测量的目的是推断出“身体”的步数和 “化学”步骤决定了速率，并探测到了 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 中间体将被用来确定本征同位素效应 用于脱羧基步骤和准确的自由能图 将会被建造。与细胞内酶的相互作用 将研究类底物抑制剂HCOOH和CO(COOH)2 动力学和溶剂同位素效应。乳酸盐 嗜冷性、中温性和嗜热性脱氢酶 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变化的方法 从热适应中反映在过渡状态 结构和各个步骤的相对大鼠。溶剂 同位素效应将探讨亲电性的重要性 氢化物转移和底物同位素的催化作用 隧道的作用。嗜热酶的突变体也会 接受检查。这些研究的结果最终应该是 在设计效应器以抑制或激活酶方面很有用。

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.