STRUCTURE AND MECHANISM OF YEAST PYRUVATE DECARBOXYLASE

这项提案涉及对剩余的结构和 丙酮酸脱羧酶的机制问题(PDC，E.C.4.1.1.1)， 可能是最简单的需要硫胺素的非氧化脱羧酶 二磷酸(ThDP)，维生素B1辅酶，具有基础意义 在人类新陈代谢中)。酿酒用酵母酶最近被提纯为 完全活性的α4和β4同分异构体，并且α4的结构是 由匹兹堡退伍军人医院的PI和合作者结晶 指出有可能成功解析到至少2.4埃。 根据已经从3埃地图上积累的证据 酶，这是第一次对这一大群人的代表 α-酮酸脱羧酶，合理的实验可以设计成 探针法：A.酶结合辅酶在无菌条件下的构象 底物以及三种共价底物-辅酶复合体中 在化学类比的基础上被援引；B.在 两个芳香环(噻唑)底物的不存在和存在 和4-氨基嘧啶)，并检验两个环(不是 只有噻唑)是催化的参与者；C. 辅酶及其周围氨基酸在催化中的作用； 以及D.丙酮酰胺，一种非脱羧基底物的作用 能够将酶从低水平转换为高水平的替代物 活动形式上、结构上和机制上。最突出的是 解决这些问题的工具是：a.x射线结晶学 方法(这部分研究是与Furey合作进行的， 萨克斯和同事在退伍军人医院生物晶体实验室/大学。的 B.发现的氨基酸的定点突变 基于x射线结晶学的催化和调节中心 机理信息；C.化学和化学的进一步阐明 ThDP底物之一的烯胺的酶结合环境 PDC上的共价化合物；D.ThDP上的多核磁共振 与PDC结合，在C2，NL‘和N4’原子上标记ThDP以提供 有关电离状态和电荷密度的信息 在催化序列中的辅酶在没有和在 监管机构在场；5.基于x射线坐标的建模 帮助改进机械模型，帮助设计更有洞察力 实验，并帮助构建两个 与PDC具有很高序列同源性的酶(丙酮酸氧化酶和 乙酰乳酸合成酶)还没有结晶，并且 通过丙酮酸脱羧基作用具有相同的机制，但不同 自那以后意义重大。毫无疑问，要解决的根本问题是 由拟议的研究解决，将具有深远的兴趣和 对从事硫胺素研究的许多其他研究小组具有重要意义 环球网。

This proposal is concerned with delineation of remaining structural and mechanistic questions on pyruvate decarboxylase (PDC, E.C. 4. 1. 1. 1), perhaps the simplest nonoxidative decarboxylase requiring thiamin diphosphate (ThDP, the vitamin B1 coenzyme, of fundamental significance in human metabolism). The brewer' yeast enzyme was recently purified as fully active alpha4 and beta4 homotetramers and the alpha4 structure was crystallized by the PI and collaborators at the VA Hospital in Pittsburgh pointing to the likely success of resolution to at least 2.4 angstroms. With the evidence already accumulated from the 3 angstroms map of the enzyme, for the first time on this representative of a large group of alpha-keto acid decarboxylases, rational experiments can be designed to probe: a. the conformation of the enzyme-bound coenzyme in the absence of substrate, as well as in the three covalent substrate-coenzyme complexes invoked, based on chemical analogy; b. the chemical behavior in the absence and presence of substrate of the two aromatic rings (thiazolium and 4-aminopyrimidine), and test of the hypothesis that both rings ( not only the thiazolium) are participants in catalysis; c. the environment of the coenzyme and the function of amino acids surrounding it in catalysis; and d. the effect of pyruvamide, a nondecarboxylatable substrate surrogate that is capable of shifting the enzyme from a low to a high activity form, on structure and mechanism. Most prominent among the tools to address these questions will be : a. x-ray crystallographic methods (this part of the research is a collaborative effort with Furey, Sax and coworkers at the VA Hospital Biocrystallography Lab/ Univ. of Pittsburgh) ; b. site-directed mutagenesis of amino acids found near the catalytic and regulatory sites based on the x-ray crystallographic and mechanistic information; c. further elucidation of the chemistry and enzyme-bound environment of the enamine, one of the three ThDP-substrate covalent complexes on PDC; d. multinuclear magnetic resonance on ThDP bound to PDC, with ThDP labelled at the C2, Nl' and N4' atoms to provide information concerning the state of ionization, and the charge densities of the coenzyme during the catalytic sequence in the absence and in the presence of regulators;. and 5. modeling based on the x-ray coordinates to help refine mechanistic models, to help design even more insightful experiments, and to help construct three dimensional models of two enzymes with very high sequence homology to PDC's (pyruvate oxidase and acetolactate synthetase) that have not been crystallized yet, and that have identical mechanisms through pyruvate decarboxylation, but diverge significantly thereafter. Undoubtedly, the fundamental questions to be resolved by the proposed research, will be of profound interest and significance to many other research groups working on thiamin around the globe.