A New Transient Kinetic Solvent Isotope Effect Approach

一种新的瞬态动力学溶剂同位素效应方法

• 批准号: 6847905
• 负责人: Harvey F. Fisher
• 金额: $ 15.34万
• 依托单位: MIDWEST VETERANS' BIOMEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6847905
• 关键词: X ray crystallography; chemical kinetics; computer simulation; deuterium; enzyme mechanism; glutamate dehydrogenase; hydrogen bond; protein structure; solvents

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.

描述（由申请人提供）：我们的长期目标是将我们对酶催化基本性质的知识扩展到比目前可用的实验方法更基础的水平。通常所写的详细酶机制的一个主要组成部分是在反应进行的各个步骤中，与酶的不同官能团和反应物相结合的质子和氢键的顺序洗牌。这种质子位移通常是从动力学和结构信息结合化学直觉的论证中推断出来的。这项提议的目的是发展并开始应用一种新的方法，在这种方法中，可以直接检测到先前确定的单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：我们展示了在酶反应中产生单个复合物的瞬态时间过程的能力；2.开发并实验验证了一种新的严格的瞬态KIE随时间行为理论，该理论可以为每个反应步骤提供有效的内在KIE。这种方法产生的结果与目前使用的结果有很大不同。我们将在牛肝脏l -谷氨酸脱氢酶（blGDH）的反应中开发和测试这种新方法。所采用的基本方法是在H2O和D2O溶剂中对反应进行多波长瞬态动力学测量。这类研究将在以下几种系统上进行：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。一些特定于个别债券建立或破坏事件的信号。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所测试的假设。我们的解释的有效性将反过来通过应用我们的瞬态溶剂KIE方法来测试一些相关的非酶反应，这些反应的详细机制已经建立。