STRUCTURE AND MECHANISM OF YEAST PYRUVATE DECARBOXYLASE

描述：这项研究的总体目标是大大提高 我们对A的结构-功能关系和机制的认识 对基础代谢至关重要的一类酶，硫胺素 依赖二磷酸的2-氧酸脱羧酶。它的X射线结构是 丙酮酸脱羧酶这类丙酮酸脱羧酶的成员(PDC，在 与W.Furey大学合作。匹兹堡)，人们可以开始分配 在原子分辨率下对单个氨基酸起作用。在当前的 授权期的方法被开发来制备PDC的变体 利用分子生物学技术，酿酒酵母。 酵母PDC既受其底物的调节，又受其 辅因子硫胺素二磷酸和镁(II)。在当前的第二个时期 调控位点上的氨基酸已被指定为功能(Cys221和 他的92岁)。这两种氨基酸位于不同的结构域，它是 假设当第一个底物结合到这个轨迹上时， 信息被传输到20埃以外的催化点。 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，而且对 辅因子诱导的调节，以及对催化的影响。的目标是 今后一个时期包括以下突出问题：1.划定 应负责的区域内替换的结构性后果 用于辅因子(所谓的硫胺素二磷酸折叠)的激活； 整个底物激活途径的描绘；3.描绘 不寻常的V辅酶构象的后果；4.描绘 氨基嘧啶环被激活的机理 催化作用；5.描述其功能和化学性质。 活性中心附近潜在的通用酸碱催化剂。工具有 提出了从野生型和变异型的X射线结晶学 酶，到稳态和稳态前的动力学，到各种 光谱学方法，帮助分配特定的功能，以执行 机理中对所有氨基酸的化学转化 牵涉到调节和催化。公安局认为，在 在接下来的几年里，终于有可能解释10E12倍 蛋白质提供的速度加速并获得亲密的 了解酶结合硫胺素是如何发挥其功能的。

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.