DIRECT OBSERVATION OF ENZYMATIC CATALYTIC STRATEGIES

直接观察酶催化策略

基本信息

批准号：
2177166
负责人：
LINDA C. KURZ
金额：
$ 16.85万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-09-27 至 1999-06-30
项目状态：
已结题

项目摘要

The long term goal of our proposed research is to understand at the molecular level how enzymes achieve their extraordinary catalytic efficiencies. We seek to do this through direct observation of enzyme- bound ligands (substrates, intermediates, and transition-state analogs). We will use techniques (NMR, IR Spectroscopies) sensitive to local bond distortions, electronic environment, protonation state and other factors likely to reflect the enzyme's catalytic strategies. We seek to do this through observations of the effects of perturbations (pH, temperature, structure of enzyme and ligand) on the standard free energies of enzyme- bound substrates, intermediates, transition-states and analogs of those states. We will use techniques (various spectroscopic titrations, calorimetry, transient kinetics) appropriate for the measurement of equilibrium and rate constants. We will make quantitative comparisons (linear free energy relationships) with the effects of these same perturbations on well-understood model systems leading to an understanding of the molecular basis of those interactions between substrate and enzyme which lead to efficient catalysis. We will focus on two groups of enzymes; the Claisen enzymes (citrate synthase) and the nucleoside aminohydrolases (adenosine deaminase). The Claisen enzymes are prominent in pathways requiring carbon-carbon bond formation in the biosynthesis of fats and cholesterol as well in the energy-yielding pathways of the tricarboxylic acid cycle and the glyoxylate cycle. These enzymes operate through activation of a substrate carbonyl together with the stabilization of an activated form of the acylthioester substrate. Enzymes which catalyze amino-nucleoside(tide) hydrolysis have more varied but no less vital roles. Adenosine deaminase is necessary to the integrity of the immune response. Other enzymes of this class participate in salvage pathways of purines and pyrimidines or are involved in the maintenance of cellular energy balance. These enzymes operate through stabilization of tetrahedral intermediates, a catalytic strategy used by several other important enzyme groups. The necessary groundwork is near completion and new results are rapidly accumulating. In previous work, we have identified catalytic strategies used by the two enzymes. We have developed probes to specifically detect and quantify the effects of perturbations on the catalytic apparatus; we have prepared enzymes with single- and multiple- site changes. We are now prepared to characterize these mutants and to continue our search for additional strategies in both groups. An understanding of how enzymes work is necessary to understand at the most fundamental level the metabolic processes in normal and disease states.

我们拟议的研究的长期目标是在分子水平酶如何实现其非凡催化效率。我们试图通过直接观察酶来做到这一点。结合配体（底物，中间体和过渡状态类似物）。我们将使用对局部键敏感的技术（NMR，IR光谱镜）扭曲，电子环境，质子化状态和其他因素可能反映了酶的催化策略。我们寻求这样做通过观察扰动的影响（pH，温度，酶和配体的结构）在酶的标准自由能上绑定的底物，中间体，过渡状态和类似物国家。我们将使用技术（各种光谱滴定，量热法，瞬态动力学）适用于测量平衡和速率常数。我们将进行定量比较（线性自由能关系）与这些相同的影响对众所周知的模型系统的扰动，导致理解底物与酶之间这些相互作用的分子基础这导致有效的催化。我们将专注于两组酶。 claisen酶（柠檬酸盐合酶）和核苷氨基氢酶（腺苷脱氨酶）。 Claisen酶在途径中很突出在脂肪的生物合成中需要形成碳 - 碳键胆固醇以及三羧基的能量赋予途径酸性循环和乙氧基循环。这些酶通过粘液羰基的激活以及稳定酰基三心细胞底物的活化形式。催化的酶氨基核苷（潮汐）水解的多种多样，但至关重要角色。腺苷脱氨酶对于免疫的完整性是必不可少的回复。该类的其他酶参与了嘌呤和嘧啶或参与细胞的维持能量平衡。这些酶是通过稳定四面体而运行的中间体，另一种其他重要酶使用的催化策略组。必要的基础工作即将完成，新结果是迅速积累。在以前的工作中，我们已经确定了催化两种酶使用的策略。我们已经开发了探针明确检测和量化扰动对催化装置；我们已经准备了单次和多重的酶站点变化。我们现在准备表征这些突变体，并继续我们在两组中寻找其他策略。一个了解酶的工作方式最多需要理解基本水平正常和疾病状态的代谢过程。