Chemical Mechanisms of Biosynthesis

生物合成的化学机制

基本信息

批准号：
6910803
负责人：
RICHARD VANCE WOLFENDEN
金额：
$ 33.49万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-07-01 至 2008-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6910803
关键词：
active sites adenosine triphosphate aldehydes chemical condensation decarboxylases enzyme activity enzyme mechanism enzyme structure enzyme substrate complex fungal proteins mass spectrometry molecular biology phosphorylation point mutation protein binding protonation thermodynamics

项目摘要

DESCRIPTION (provided by applicant): A serious challenge faced by an enzyme's active site is to avoid tight binding of the altered substrate in the ground state, as contrasted with the active site's very high affinity for the altered structure in the transition state. In the first part of this project, modified nucleotides, point mutations and structure determination will be used to investigate the binding properties of yeast orotidine 5'-phosphate decarboxylase, an unusually powerful catalyst. The principal goal of these experiments is to investigate the extent to which steric versus electrostatic interactions may be responsible for the instability of the ground state enzyme-substrate complex. In the second part of this project, structural methods, including mass spectrometry, will be used to investigate the extent to which the shortcomings of transition state analogues may arise from their possible tendency to trap water molecules at the active site, a possibility that is consistent with the observed thermodynamics of binding of transition states and transition state analogue inhibitors by E. coil cytidine deaminase. In the third part of this project, the rates of several uncatalyzed 2-substrate reactions, including aldol condensation, alcohol dehydrogenation, phosphorylation of alcohols by ATP, and peptidyl transfer, will be determined as a function of changing temperature. The goal of these experiments is to obtain benchmarks for comparison with rate constants to be obtained for the corresponding enzyme reactions, to estimate the affinity expected of an ideal bisubstrate analogue inhibitor of enzymes that catalyze each of these reactions, and to determine the extent to which the thermodynamics of activation and transition state binding by these enzymes may differ from those of enzymes catalyzing one-substrate and hydrolytic reactions. The hypotheses that will be tested in each of the three parts of this project have a significant bearing on the design of drugs and other enzyme inhibitors of practical interest.

描述（由申请人提供）：酶的活性位点面临的严峻挑战是避免基态中改变的底物的紧密结合，这与活性位点对过渡态中改变的结构的非常高的亲和力形成对比。在该项目的第一部分中，修饰核苷酸、点突变和结构测定将用于研究酵母乳清苷5'-磷酸脱羧酶（一种异常强大的催化剂）的结合特性。这些实验的主要目标是研究空间相互作用与静电相互作用在多大程度上可能导致基态酶-底物复合物的不稳定。在该项目的第二部分中，将使用包括质谱在内的结构方法来研究过渡态类似物的缺点在多大程度上可能是由于它们可能在活性位点捕获水分子的倾向而产生的，这种可能性是一致的观察到大肠杆菌胞苷脱氨酶与过渡态和过渡态类似物抑制剂结合的热力学。在该项目的第三部分中，几种非催化 2-底物反应的速率，包括醛醇缩合、醇脱氢、ATP 醇磷酸化和肽基转移，将被确定为温度变化的函数。这些实验的目的是获得与相应酶反应所获得的速率常数进行比较的基准，估计催化每个反应的酶的理想双底物类似物抑制剂的预期亲和力，并确定其作用的程度。这些酶的活化和过渡态结合的热力学可能不同于催化单底物和水解反应的酶的热力学。将在该项目的三个部分中进行测试的假设对于药物和其他具有实际意义的酶抑制剂的设计具有重大影响。