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知识扩展到比目前可用的实验方法所允许的更基本的水平。通常所写的详细的酶机理的一个主要组成部分是随着反应的各个步骤的进行，氢键键合到酶的不同功能基团的质子及其反应物种类的顺序洗牌。这样的质子移动通常是从基于动力学和结构信息的论点结合化学直觉推断出来的。这项提议的目的是开发并开始应用一种新的方法，在这种方法中，可以直接探测到先前确定的个别反应步骤中的特定质子的运动。这一方法是基于与本实验室最近的工作相结合的两个不同的发展：1.我们证明了在酶反应中产生单个络合物的瞬时时间过程的能力；2.发展和实验验证了瞬时Kie的时间依赖行为的新的严格理论，该理论为每个反应步骤产生有效的固有Kie。这种方法产生的结果与目前使用的结果有很大不同。我们将在牛肝L-谷氨酸脱氢酶的反应中开发和测试这一新方法。所采用的基本方法是在水和D2O溶剂中对反应进行多波长暂态动力学测量。这样的研究将在以下几类系统上进行：1.各种条件下的全反应时间过程；2.几个已知的部分反应L和3.个别成键或断键事件特有的信号。基于结晶学和计算机模拟结构的详细反应机理将作为要由我们的结果检验的假设。我们的解释的有效性将通过我们的暂态溶剂KIE方法应用于一些相关的非酶反应来检验，这些反应的详细机制已经很好地建立起来了。

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.