STRUCTURE AND MECHANISMS OF ACTION OF ENZYMES

本实验室的长期目标是确定 酶催化反应的机制，并以更普遍的方式 以确定分子水平上生物相互作用的性质， 水平 除此之外，我们现在还试图获得一些理解 催化过程的能量学， 在各个步骤的能量学和化学反应之间 发生在这些步骤中。 多年来，我们一直专注于 谷氨酸脱氢酶系统的研究提供了广泛的 各种各样的现象与追求这样的研究。 他已于去年 列出了形成的九种主要复合物的发生顺序 沿着反应时间过程，我们现在转向详细的研究。 这些络合物相互转化中的化学事件。 在 在这项建议中，我们把注意力集中在以下两个关键领域： 反应顺序：1）羰基的作用是什么？ α-酮戊二酸，以及在哪一点上所需的水 分子进入反应过程？ 这一目标将通过以下方式加以探讨： 使用缺乏羰基的类似物， 问题研究 2)什么是事件和复合之前和伴随 氢化物转移步骤？ 这将通过一项研究来探讨， 相关的模型反应，并通过对早期瞬态的平行研究 步 后一项研究将包括冷冻酶学和替代方法。 动力学的方法来研究瞬变，这可能有一些 应用于一般酶的研究。 最后，我们将尝试 将最近发现的温度依赖平衡 游离酶的两种异构形式之间的水合作用的变化， (or)在许多复合物中观察到的大的负Δ Cp 吡啶核苷酸转移酶。 这些最初的实验探测器 是在这一领域作出认真努力的前奏， 部分，以确定是否，正如詹克斯所建议的，部分基板 结合能可被引导以驱动随后的催化步骤，并且 若有，是如何完成的。

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.