CATALYTIC ACTIVITIES OF TARTRATE DEHYDROGENASE

该提案概述了一种实验方法，以了解 细菌酒石酸盐酶的详细化学机制 脱氢酶（TDH）。 TDH是一种不寻常的酶， 三种不同的底物催化不同的化学反应 每一个人。 催化反应中的速率控制步骤 将通过稳态动力学研究确定， 中间体分配实验和同位素效应研究。 描述了实验，这将允许计算的内在 同位素效应的酶促反应，从过渡 国家结构，因此，起源的催化能力，可以是 推断。 底物类似物研究将探测分子决定因素 每一个反应的能量成本，以及随后一个反应的能量成本 路与另一条路 将克隆编码TDH的基因， 随机诱变并表达，以确定蛋白质是否可以 它能催化最慢的反应 由野生型酶催化。 突变蛋白质将 动力学特征，以确定其增加的基础 效率，并确定是否有能力催化其他 反应已经改变。 这些研究应该确定酶促反应的哪些部分是 最容易优化，各种酶底物的作用 相互作用决定了催化反应的结果。 了解酶促反应速率的机制 是有限的，底物反应性的机制是 如果酶被合理地操纵， 在生物技术中使用的方式，并被理解为有效的目标 药物设计。 TDH是进行此类研究的理想系统 因为它似乎是一种微妙的平衡， 不同的反应，因此，在其相互作用的微妙变化， 底物对催化反应的结果有显著的影响。 反应

This proposal outlines an experimental approach to understanding the detailed chemical mechanisms of the bacterial enzyme tartrate dehydrogenase (TDH). TDH is an unusual enzyme in that it recognizes three different substrates and catalyzes a different chemical reaction with each one. The rate-determining steps in the catalytic reactions will be determined by steady-state kinetic studies, intermediate-partitioning experiments, and isotope effect studies. Experiments are described which will allow calculation of the intrinsic isotope effects on the enzymatic reactions, from which the transition state structures, and thus the origin of the catalytic power, can be inferred. Substrate analog studies will probe the molecular determinants of each reaction, and the energetic costs of following one reaction pathway versus another. The gene encoding TDH will be cloned, and randomly mutagenized and expressed to determine whether a protein can be created which has enhanced ability to catalyze the slowest reaction catalyzed by the wild-type enzyme. The mutant proteins will be characterized kinetically to determine the basis for their increased efficiency, and to determine whether the ability to catalyze the other reactions has changed. Such studies should identify which segments of an enzymatic reaction are most easily optimized, and the roles that various enzyme-substrate interactions play in determining the outcome of the catalytic reaction. Understanding the mechanisms by which the rates of enzymatic reactions are limited, and the mechanisms by which substrate reactivity is controlled, is necessary if enzymes are to be manipulated in a rational manner for use in biotechnology and understood as targets for efficient design of pharmaceuticals. TDH is an ideal system for such studies because it appears to be finely balanced as a catalyst for three different reactions, so that subtle changes in its interactions with the substrates have a dramatic effect on the outcome of the catalytic reaction.