PYRUVATE DECARBOXYLASE STRUCTURE-FUNCTION RELATIONSHIPS

描述：本研究的总体目标是 扩展了我们对一类结构-功能关系的知识， 对基本代谢至关重要的酶;硫胺素 二磷酸依赖性α-酮酸脱羧酶。 尽管他们 在生物化学和多年的研究中广泛的重要性，即使是最 基本的结构信息只是最近才成为任何 这个家庭的成员，许多问题仍然没有答案， 催化和调节机制。 现在的目标是利用和 扩展最近获得的基本结构信息， 代表性的酶丙酮酸脱羧酶（PDC），通过提供详细的 关于结构变化的信息， 变构激活剂，从抑制剂相互作用的催化和 调控位点，并从重要的单残基突变引入 在调节和催化位点的位置。 完成这项工作的方法是 通过测定和分析全酶复合物的三维结构， 通过单晶X-射线衍射方法， 与天然PDC的结构进行比较。 具体目标是： (1)确定并分析了复合材料的结构， 变构激活剂和底物替代物酮丙二酸酯和阿托伐米胺; (2)在复合物之间进行详细的结构比较， 活化剂和天然PDC结构，并测试几个假设 关于变构激活机制;（3）确定结构 由在催化剂中引入的单残基突变引起的效应 位点，并影响酶活性，并测试一个假设 辅因子稳定的酶;（4）以确定结构的影响 由于在调节位点引入的单残基突变而导致 和影响变构活化过程;（5）检查 在沿着 通过分析具有催化位点的络合物的反应途径 抑制剂乙醛酸和乙酰次膦酸甲酯;（6）检测 与调控位点反应后的全酶结构 抑制剂，如CPB;（7）进行详细的结构比较， 与抑制剂和天然PDC结构的复合物;（8）以获得 详细的，高分辨率的三斜形PDC结构分析。

DESCRIPTION: The overall objective of this research is to substantially extend our knowledge of structure-function relationships in a class of enzymes of fundamental importance to basic metabolism; the thiamin disphosphate dependent alpha-keto acid decarboxylases. Despite their widespread importance in biochemistry and many years of study, even the most basic structural information has only very recently become available for any member of this family, and numerous questions remain unanswered regarding the catalytic and regulatory mechanisms. The goal now is to exploit and expand upon the elementary structural information recently obtained for the representative enzyme pyruvate decarboxylase (PDC), by providing detailed information about structural changes resulting from interaction with allosteric activators, from inhibitors interacting at the catalytic and regulatory sites, and from single residue mutations introduced at important locations in both the regulatory and catalytic sites. This will be achieved by determining and analyzing the 3D structures of holoenzyme complexes and mutant enzymes via single crystal x-ray diffraction methods, and by comparing the structures with that for native PDC. The specific aims are: (1) to determine and analyze the structure of PDC when complexed with the allosteric activators and substrate surrogates ketomalonate and pyruvamide; (2) to make detailed structural comparisons between complexes with activators and the native PDC structure, and to test several hypotheses regarding the allosteric activation mechanism; (3) to identify structural effects resulting from single residue mutations introduced in the catalytic site and which affect enzyme activity, and to test a hypothesis regarding cofactor stabilization by the enzyme; (4) to identify structural effects resulting from single residue mutations introduced in the regulatory site and which affect the allosteric activation process; (5) to examine the structure of the holoenzyme when frozen at intermediate stages along the reaction pathway by analysis of complexes with the catalytic site directed inhibitors glyoxalic acid and methyl acetylphosphinate; (6) to examine the structure of the holoenzyme after reaction with regulatory site directed inhibitors such as CPBs; (7) to make detailed structural comparisons between complexes with the inhibitors and the native PDC structure; (8) to obtain a detailed, high resolution structural analysis of the triclinic form of PDC.