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: 7009330
• 负责人: Harvey F. Fisher
• 金额: $ 14.98万
• 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 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7009330
• 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允许的更基础的水平。通常所写的详细酶机制的一个主要组成部分是，当反应通过其各个步骤进行时，与酶及其反应物种类的不同官能团氢键合的质子的顺序改组。这种质子位移通常是根据动力学和结构信息与化学直觉相结合的论证推断出来的。该提案的目的是开发并开始应用一种新方法，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。这种方法基于该实验室最近工作的两种不同进展的结合： 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. 瞬态 KIE 的时间依赖性行为的新颖严格理论的开发和实验验证，该理论为每个反应步骤产生有效的内在 KIE。该方法产生的结果与当前使用的结果有很大不同。我们将在经过充分研究的牛肝 L-谷氨酸脱氢酶 (blGDH) 反应上开发和测试这种新方法。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。此类研究将在以下类型的系统上进行： 1. 各种条件下的完整反应时间过程； 2. 几个已知的部分反应 L 和 3. 一些特定于单个成键或断键事件的信号。基于晶体学和计算机建模结构的详细反应机制将作为我们的结果检验的假设。我们的解释的有效性反过来将通过将我们的瞬态溶剂 KIE 方法应用于许多相关的非酶反应来测试，这些反应的详细机制已经明确。