ENZYME EFFECTOR DESIGN

丝氨酸蛋白酶，酰胺水解酶的催化能力， 细菌、丙酮酸脱羧酶和乳酸脱氢酶， 嗜冷菌、嗜中温菌和嗜热菌、GABA-T和可能的一些其他生物。 酶将通过测定主要和 二次氢同位素效应、碳同位素效应和溶剂效应 同位素效应 后者将由质子库存分析 将整体效应分解为个体贡献的方法 地点，只要有可能。 目的是从同位素中学习 对酶的过渡态结构的影响 反应，并比较酶的过渡态与其他 酶促反应和非酶促反应，以推断 酶正在产生催化加速作用。 同位素效应， 确定有助于限制速率的步骤的性质 在不同的条件下，也有助于显示酶如何结合联合收割机多步骤 过程以生成完整的机械序列。 对于丝氨酸蛋白酶，将用以下方法进行质子库存： 各种长度和序列的寡肽底物，以发现 耦合多质子催化机制的先决条件是什么 的酶。 基质结构变化的程度， 将研究伴随这种活化的羰基反应， 二次氘同位素效应 类似的实验， 将进行天冬酰胺酶和天冬酰胺酶的底物。 为 丙酮酸脱羧酶，pH和温度的影响，直接 测量的C-13效应将用于发现这些变量如何影响 脱羧和其他步骤对速率限制的贡献。 溶剂同位素效应将类似地用于探测以下物质的作用： 质子转移的机制。 对于乳酸脱氢酶， 一次和二次氢效应应该揭示了 氨基酸序列，伴随着遗传适应的 细菌对不同温度制度的反应反映在结构上 活化复合物和不同步骤的相对贡献 速率限制，无论是在最佳和非最佳条件下，每一个 酵素 水解反应溶剂同位素效应的温度依赖性 酶也将测试是否需要隧道模型的酶 酸碱催化

The catalytic power of serine proteases, amidohydrolases from various bacteria, pyruvate decarboxylase, and lactate dehydrogenases from a psychrophile, a mesophile and a thermophile, GABA-T and possibly some other enzymes will be investigated through the determination of primary and secondary hydrogen isotope effects, carbon isotope effects, and solvent isotope effects. The latter will be analyzed by the proton-inventory method to dissect the overall effect into contributions from individual sites, wherever possible. The objective is to learn from the isotope effects about the structure of the transition states for the enzymic reactions and to compare the enzymic transition states with those for other enzymic reactions and non-enzymic reactions, in order to deduce how the enzyme is producing its catalytic acceleration. Isotope effects, by defining the nature of the steps which contribute to limiting the rate under various conditions, also help to show how enzymes combine multistep processes to generate a complete mechanistic sequence. For the serine proteases, proton inventories will be conducted with oligopeptide substrates of various length and sequence in order to discover what the prerequisites are for coupling the multiproton catalytic machinery of the enzyme. The degree to which substrate structural changes at the reacting carbonyl accompany this activation will be investigated with secondary deuterium isotope effects. Similar experiments with various substrates of asparaginases and glutaminases will be carried out. For pyruvate decarboxylase, the effects of pH and temperature on directly measured C-13 effects will be used to discover how these variables affect the contribution of decarboxylation and other steps to rate limitation. Solvent isotope effects will be similarly used to probe the role of proton-transfer components of the mechanism. For lactate dehydrogenases, primary and secondary hydrogen effects should reveal how the changes in amino-acid sequence which have accompanied the genetic adaptation of the bacteria to different temperature regimes are reflected in the structure of the activated complexes and the relative contributions of different steps to rate limitation, both under optimal and non-optimal conditions for each enzyme. Temperature dependences of solvent isotope effects for hydrolytic enzymes will also test whether a tunneling model is required for enzymic acid-base catalysis.