STRUCTURE AND MECHANISM OF YEAST PYRUVATE DECARBOXYLASE

描述：本研究的总体目标是大大提高 我们的知识的结构功能关系和机制的一个 硫胺素：一类对基本代谢至关重要的酶，硫胺素 二磷酸依赖性2-酮酸脱羧酶。 其X射线结构 这类丙酮酸脱羧酶（PDC）的成员，在一个实施方案中解决。 与W合作。Furey，匹兹堡大学），人们可以开始分配 在原子分辨率下对单个氨基酸起作用。 于本 开发了授权期方法，以从 酵母酿酒酵母，利用分子生物学技术。 酵母PDC受其底物和其代谢产物的调节。 辅因子二磷酸硫胺素和Mg（II）。 在本报告所述期间， 调控位点的氨基酸已被指定功能（Cys 221和 His92）。 这两种氨基酸在不同的结构域上， 假设当第一底物与该位点结合时， 信息被传送到20埃以外的催化位点。 此外，催化中心的几个氨基酸被鉴定为 不仅对辅因子结合有重大影响， 辅因子诱导的调节，以及催化。 下组织生产的目标 今后一个时期突出的问题有：1. 划定 的结构性后果的替代在该地区负责 用于通过辅因子激活（所谓的二磷酸硫胺素折叠）; 2. 整个底物活化途径的描绘; 3. 划定 不寻常的V辅酶构象的后果; 4. 划定 负责氨基嘧啶环活化的机制 催化;和5. 功能和化学性质的描述 活性中心附近潜在的一般酸/碱催化剂。 工具 从野生型和变异型的X射线晶体学 酶，稳态和前稳态动力学，各种 光谱方法，以帮助分配特定的功能， 所有氨基酸的化学转化机制 参与调节和催化。 PI认为，在 未来几年，最终将有可能解释10 E12倍 蛋白质提供的速率加速， 了解酶结合硫胺素如何发挥其功能。

DESCRIPTION: The overall objective of this research is to very much enhance our knowledge of the structure-function relationships and mechanism of a class of enzymes of fundamental importance to basic metabolism, the thiamin diphosphate-dependent 2-oxoacid decarboxylases. With the X-ray structure of a member of this class pyruvate decarboxylase (PDC, solved in a collaboration with W. Furey, Univ. of Pittsburgh), one can begin to assign function to individual amino acids at atomic resolution. During the current grant period methods were developed to prepare variants of PDC from the yeast Saccharomyces cerevisiae, using techniques in molecular biology. Yeast PDC is subject to regulation both by its substrate and by its cofactors thiamin diphosphate and Mg(II). During the current period two amino acids at the regulatory site have been assigned function (Cys221 and His92). These two amino acids are on different domains and it is hypothesized that when the first substrate binds to this locus, the information is transmitted to the catalytic site more than 20 Angstrom away. In addition, several amino acids in the catalytic center were identified as having a major impact not only on cofactor binding, but also on cofactor-induced regulation, as well as on catalysis. The goals for the coming period include the following outstanding problems: 1. delineation of the structural consequences of substitutions in the region responsible for activation by cofactors (the so-called thiamin diphosphate fold); 2. delineation of the entire substrate activation pathway; 3. delineation of the consequences of the unusual V coenzyme conformation; 4. delineation of the mechanism responsible for activation of the aminopyrimidine ring in catalysis; and 5. delineation of the function and chemical properties of potential general acid/base catalysts near the active center. Tools are being proposed, ranging from X-ray crystallography of wild-type and variant enzymes, to steady-state and pre-steady-state kinetics, to a variety of spectroscopic methods to help assign specific function for carrying out the chemical transformations in the mechanism to all of the amino acids implicated in regulation and catalysis. The PI believes that during the coming few years it will finally be possible to account for the 10E12-fold rate acceleration that the protein provides and to gain an intimate understanding of how enzyme-bound thiamin performs its function.