ENZYME EFFECTOR DESIGN

不同来源的丝氨酸蛋白酶、氨基水解酶的催化能力 细菌、丙酮酸脱羧酶和乳酸脱氢酶 嗜冷者，嗜中者和嗜热者，GABA-T和可能的其他一些 酶的研究将通过测定初级和 二次氢同位素效应、碳同位素效应和溶剂 同位素效应。后者将通过质子清单进行分析 将整体效应分解为个人贡献的方法 地点，只要有可能。我们的目标是从同位素中学习 酶过渡态结构对酶活性的影响 反应，并将酶过渡态与其他 酶反应和非酶反应，以推断如何 酶正在产生催化加速作用。同位素效应，由 定义有助于限制费率的步骤的性质 在各种条件下，也有助于展示酶是如何结合多步的 生成一个完整的机械序列的过程。 对于丝氨酸蛋白酶，质子库存将与 不同长度和序列的寡肽底物以发现 耦合多质子催化装置的前提条件是什么？ 酶的活性。衬底结构变化的程度 伴随着这一活化反应的羰基将被用 二次氚同位素效应。类似的实验有不同的 将进行天冬酰胺酶和谷氨酰胺酶底物的研究。为 丙酮酸脱羧酶对pH和温度的直接影响 测量的C-13效应将被用来发现这些变量如何影响 脱羧基和其他步骤对心率限制的贡献。 溶剂的同位素效应将同样被用来探索 该机构的质子转移部件。对于乳酸脱氢酶， 原氢效应和次氢效应应该揭示 伴随着该基因的遗传适应的氨基酸序列 细菌对不同温度制度的影响体现在结构上 活化络合物及其在不同步骤中的相对贡献 在最优和非最优条件下进行速率限制 酵素。水解物的溶剂同位素效应与温度的关系 酶还将测试酶是否需要隧道模型 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.