A New Transient Kinetic Solvent Isotope Effect Approach

描述（由申请人提供）：我们的长期目标是将我们对酶催化的基本性质的了解扩展到比目前可用的实验方法所允许的更基本的水平。详细的酶机制的一个主要组成部分，通常写的是一个连续的改组质子氢键的不同功能基团的酶和它的反应物物种的反应进行通过其各个步骤。这种质子位移通常是从基于动力学和结构信息结合化学直觉的论点中推断出来的。该建议的目的是开发并开始应用一种新的方法，其中可以直接检测先前确定的各个反应步骤中特定质子的运动。这种方法是基于本实验室最近工作的两个不同发展的结合：1。我们证明了在酶反应中产生单个复合物的瞬态时间过程的能力; 2.， 发展和实验验证了一种新的严格的理论的瞬态KIE的时间依赖性的行为，产生有效的内在KIE的每一个反应步骤。这种方法产生的结果与目前使用的结果大不相同。我们将在牛肝L-谷氨酸脱氢酶（blGDH）的充分研究反应上开发和测试这种新方法。所采用的基本方法是在H2O和D2 O溶剂中的反应的多波长瞬态动力学测量。这些研究将在以下几种系统上进行：1。各种条件下的全反应时程; 2.几个已知的部分反应L和3。某些信号对于单个键形成或键断裂事件是特定的。基于晶体学和计算机模拟结构的详细反应机制将作为我们的结果进行测试的假设。我们的解释的有效性将反过来测试我们的瞬态溶剂KIE方法的应用程序的一些相关的非酶促反应，其详细的机制是完善的。

DESCRIPTION (provided by applicant): Our long range goal is to extend our knowledge of the basic nature of enzymatic catalysis to a more fundamental level than currently available experimental approaches permit. A major component of detailed enzyme mechanisms as customarily written is a sequential shuffling of protons hydrogen bonded to varying functional groups of the enzyme and its reactant species as the reaction proceeds through its various steps. Such proton shifts are usually inferred from arguments based on kinetic and structural information combined with chemical intuition. The aims of this proposal are to develop and to begin to apply a new approach in which the movement of specific protons in previously identified individual reaction steps can be detected directly. This approach is based on the combining of two different developments from the recent work of this laboratory: 1. Our demonstrated ability to produce transient-state time courses of individual complexes in an enzyme reaction; 2., the development and experimental verification of a novel rigorous theory of the time-dependent behavior of transient-state KIE's which yields valid intrinsic KIE's for each reaction step. This method produces results which differ substantially from those currently in use. We will develop and test this new approach on the well studied reaction of bovine liver L-glutamate dehydrogenase (blGDH). The basic method employed is the multiwavelength transient-state kinetic measurement of the reaction in both H2O and D2O solvents. Such studies will be carried out on the following kinds of systems: 1. the full reaction time course under various conditions; 2. several known partial reactionsL and 3. some signals which are specific to an individual bond-making or bond-breaking event. A detailed reaction mechanism based on crystallographic and computer modeled structures will serve as the hypothesis to be tested by our results. The validity of our interpretations will in turn be tested by application of our transient-state solvent KIE approach to a number of relevant non-enzymatic reactions whose detailed mechanisms are well established.