STRUCTURE AND MECHANISMS OF ACTION OF ENZYMES

这个实验室的长期目标是确定 酶催化反应的机制，以及更一般的方式 在分子水平上确定生物相互作用的性质 水平。对于这些，我们现在增加了一项尝试，以获得一些理解 对催化过程的能量学的认识和了解 在单个步骤的能量学和化学事件之间 在这些步骤中发生的。多年来，我们一直专注于 谷氨酸脱氢酶系统的研究 用来进行这种研究的各种现象。已经有了 圈定了形成的九大杂岩的产状顺序 沿着反应时间的路线，我们现在转向详细的研究 在这些络合物的相互转化中涉及的化学事件。在……里面 这项建议我们将注意力集中在 反应顺序：1)羧基的作用是什么 α-酮戊二酸，以及所需的水到底在哪一点 分子进入反应过程？这一目标将通过 使用了一种不含羰基的类似物，并由各种0-1-8 学习。2)之前和伴随的事件和情结是什么 氢化物转移步骤？这一点将通过一项非常 相关的模型反应和对早期瞬变的平行研究 台阶。后一项研究将包括低温酶学和替代研究 研究瞬变的动力学方法可能有一些 一般应用于酶的研究。最后，我们将尝试 来描述最近发现的依赖温度的平衡平衡 在游离酶的两种异构体之间对水合作用和 (或)到在许多络合物中观察到的大的负DeltaCp 吡啶核苷酸脱氢酶。这些最初的实验探测器 是定义我们在这一领域的认真努力的前奏 部分用于确定是否如Jencks所建议的那样，底物的一部分 结合能可以被引导到驱动较晚的催化步骤，并且 如果是这样，它是如何实现的。

The long-range objectives of this laboratory are to determine the mechanisms by which enzymes catalyze reactions, and in a more general way to determine the nature of biological interactions at the molecular level. To these we have now added an attempt to gain some understanding of the energetics of the catalytic process and to learn the relationship between the energetics of individual steps and the chemical events occurring in those steps. For some years we have focused our investigations on the glutamic dehydrogenase system which offers a wide variety of phenomena with which to pursue such studies. Having already blocked out the order of occurrence of the nine major complexes formed along the reaction time course, we now turn to the study of the detailed chemical events involved in the interconversions of those complexes. In this proposal we focus our attention on two critical areas of the reaction sequence: 1) What is the role of the carbonyl group of Alpha-ketoglutarate, and at precisely which point does the required water molecule enter the reaction course? This aim will be explored through the use of an analog lacking the carbonyl group and by a variety of 0 1 8 studies. 2) What are the events and complexes preceding and accompanying the hydride transfer step? This will be explored by a study of very pertinent model reactions and by parallel studies on the early transient steps. This latter study will include cryoenzymology and alternative kinetic approach to the study of transients which may have some application to the study of enzymes in general. Finally, we will attempt to relate a recently discovered temperature-dependent poised equilibrium between two isomeric forms of the free enzyme to changes in hydration and (or) to the large negative DeltaCp's observed in many of the complexes of pyridine nucleotide dehydrogenases. These initial experimental probes are a prelude to the definition of a serious effort in this area on our part to determine whether, as Jencks has suggested, part of the substrate binding energy can be channeled into driving a later catalytic step, and if so, how it is accomplished.