Enzyme Structure-Function Relationships

9418479雷明顿X射线结晶学和诱变研究将对两种酶进行，来自猪和固氮菌的柠檬酸合成酶和来自小麦和酵母的丝氨酸羧基肽酶。猪、小麦和酵母酶的原子模型已经在手边，并以高分辨率进行了精炼。这些将被用作抑制剂结合研究的基础，该研究将探索催化机理的几个方面。已经设计了一些抑制剂来模拟催化途径中各个步骤的过渡态，从而提供酶的构象以及与催化途径中底物相互作用的“快照”。酵母丝氨酸羧肽酶发生突变，导致底物专一性发生很大变化，与多肽醛的结合研究将揭示突变的结构后果和底物专一性的分子基础。为了研究细菌柠檬酸合成酶是如何受NADH变构调节的，以及它们在结构上与猪的非变构调节酶有何不同，将确定固氮菌的酶的结构。这种细菌酶还将被用作诱变研究的载体，旨在探索特定活性部位氨基酸侧链的功能。其动机是，现有的许多有趣的猪酶突变体都不能结晶，因此突变的结构后果尚不清楚。这些项目有望阐明两种不同酶家族的酶机制。每种酶都会引起关于催化机制的重大问题，但仍有待了解。柠檬酸合成酶是唯一一种结构已知的能形成碳-碳键的酶。反应的第一步--碳酸的去质子化--的能量学还不是很清楚。丝氨酸羧肽酶是一种丝氨酸蛋白酶，与其他丝氨酸蛋白酶不同，它在pH 4.0-5.0时功能最佳，其结构基础也不清楚。将对猪和固氮菌的柠檬酸合成酶和小麦和酵母的丝氨酸羧基肽酶两种酶进行%%X射线结晶学和诱变研究。这些项目有望阐明这两个不同酶家族的酶机制。每种酶都有关于催化机制的重大问题，但仍有待了解。将确定固氮菌酶的结构，以便研究这些细菌酶是如何调节的，以及它们与猪酶有何不同。细菌酶也将发生突变，以探索特定活性部位氨基酸侧链的功能。酵母丝氨酸羧肽酶发生突变，导致底物专一性发生很大变化。与底物类似物的结合研究应该揭示突变变化的结构后果和底物特异性的分子基础。***

9418479 Remington X-ray crystallographic and mutagenesis studies will be done on two enzymes, citrate synthase from pig and Azotobacter and serine carboxypeptidase from wheat and yeast. Atomic models for the pig, wheat and yeast enzymes are on hand and have been refined at high resolution . These will be used as the basis for inhibitor binding studies that will probe several aspects for the catalytic mechanism. Inhibitors have been designed that mimic proposed transition states of various steps along the catalytic pathway, so as to provide "snapshots: of enzyme conformations and interactions with substrate along the catalytic pathway. The yeast serine carboxypeptidase has been mutated, resulting in greatly changed substrate specificity, and binding studies with peptide aldehydes should reveal the structural consequences of the mutational change and the molecular basis for substrate specificity. The structure of the Azotobacter enzyme will be determined in order to study how bacterial citrate synthases are allosterically regulated by NADH, and how they differ in structure from the non-allosterically regulated enzyme from pig. The bacterial enzyme will also be used as a vehicle for mutagenesis studies which will be designed to probe the functions of specific active-site amino acid side chains. The impetus for this is that most of many interesting mutants of the pig enzyme that exist cannot be crystallized, so the structural consequences of the mutations are unknown. These projects are expected to shed light on the enzymatic mechanisms for two different families of enzymes. Each enzyme poses significant problems concerning the mechanism of catalysis that remain to be understood. Citrate synthase is the only enzyme of known structure that can form a carbon-carbon bond. The energetics of the initial step in the reaction, the deprotonation of a carbon acid, are not understood. Serine carboxypeptidases are serine proteinases that function optimally at pH 4.0-5.0, unlike the other serine proteinases, and the structural basis for this is also not understood. %% X-ray crystallographic and mutagenesis studies will be done on two enzymes, citrate synthetase from pig and Azotobacter, and serine carboxypeptidase from wheat and yeast. These projects are expected to shed light on the enzymatic mechanisms for the two different families of enzymes. Each enzyme possesses significant problems concerning the mechanism of catalysis that remain to be understood. The structure of the Azotobacter enzyme will be determined in order to study how these bacterial enzymes are regulated and how they differ from the pig enzyme. The bacterial enzyme will also be mutated to probe the function of specific active-site amino acid side chains. The yeast serine carboxypeptidase has been mutated, resulting in greatly changed substrate specificity. Binding studies with substrate analogs should reveal the structural consequences of the mutational change and the molecular basis of substrate specificity. ***