PYRUVATE DECARBOXYLASE STRUCTURE-FUNCTION RELATIONSHIPS

描述：这项研究的总体目标是 在一类结构-功能关系中扩展我们的知识 对基础代谢至关重要的酶；硫胺素 依赖二磷酸的α-酮酸脱羧酶。尽管他们 在生物化学中的广泛重要性和多年的研究，甚至是最 直到最近，基本的结构信息才能用于任何 这个家庭的成员，还有许多问题没有得到回答 催化和调节机制。现在的目标是利用和 根据最近获得的基本结构信息展开 代表酶丙酮酸脱羧酶(PDC)，通过提供详细的 关于结构变化的信息，这些变化由 变构激活剂，来自在催化剂和 调节位点，以及从重要的引入的单一残基突变 在调控和催化部位的位置。这将会实现的 通过测定和分析全酶复合体的三维结构 通过单晶x射线衍射法和通过 并与原生PDC的结构进行了比较。具体目标是： (1)测定和分析PDC与PDC络合时的结构 变构激活剂和底物替代物酮丙酸和丙酮酰胺； (2)进行详细的结构对比 激活剂和本地PDC结构，并测试几个假设 关于变构活化机理的研究；(3)鉴定结构 催化剂中引入的单残基突变所产生的影响 以及影响酶活性的因素，并检验关于 用酶稳定辅因子；(4)鉴定结构效应 由调节位点中引入的单一残基突变引起 以及影响变构活化过程的因素；(5)研究 全酶在中间阶段冷冻时的结构 通过分析具有催化中心的络合物的反应路径 乙醛酸和乙酰基膦酸甲酯的抑制剂；(6)检查 与调控位点定向反应后的全酶结构 抑制剂，如多氯联苯；(7)进行详细的结构比较 与抑制剂和天然PDC结构的络合物；(8)获得 详细的、高分辨率的PDC三斜形结构分析。

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.