Structure and Mechanism of Yeast Pyruvate Decarboxylase

描述（申请人提供）：本研究的总体目标是增强我们对酵母丙酮酸脱羧酶的结构-功能关系的理解，包括酵母丙酮酸脱羧酶的机制和调控。这种酶需要二磷酸硫胺素（维生素B1辅酶），并在酒精发酵的倒数第二步使丙酮酸脱羧。申请下一阶段继续研究该酶的资金，主要目标如下：(1)使用各种光谱方法进一步研究催化残基的作用。通过使用中间分配和快速猝灭方法直接评估所有关键硫胺素二磷酸结合中间体的浓度，确定所有活性中心变异以及野生型酶的限速步骤；(2) PI及其同事提出的该酶的“功能性二聚体中活性位点交替”机制的结构起源和途径，特别是通过识别催化中心之间的信号转导途径，设计了旨在确定该酶的结构起源和途径的实验。实验旨在确定二聚体相互作用的结构起源和途径，导致x射线研究中观察到的两种不同的构象。特别是，Pl’s小组最近报告的动力学研究表明，酶有两种活性构象，一种受底物调节，另一种不受底物调节。因此，进一步描绘这两种构象的结构基础是很重要的，而那些可以结晶的构象将受到高分辨率x射线方法的影响，这是与匹兹堡大学医学院的William Furey正在进行的合作。目标1和2将在很大程度上依赖于Pl的能力，不仅可以引入任何期望的取代，而且还可以创建具有任何期望的修饰亚基组成的四聚体。该酶不仅为硫胺素依赖性酶，而且为同型四聚体酶活性中心的相互作用提供了一个杰出的范例。

DESCRIPTION (provided by applicant): The overall objective of this research is to very much enhance our understanding of structure-function relationships, including the mechanism and regulation of the yeast pyruvate decarboxylase. This enzyme requires thiamin diphosphate (the vitamin B1 coenzyme) and decarboxylates pyruvate in the penultimate step of alcohol fermentation. Funding is requested for the next period to continue research on this enzyme with the following major goals: (1) Further studies of the role of catalytic residues using a variety of spectroscopic methods. Determination of the rate-limiting steps in all active center variants, as well as wild-type enzyme, by direct assessment of the concentration of all key thiamin diphosphate-bound intermediates using intermediate partitioning and rapid-quench methods; (2) Experiments designed to identify the structural origins of and pathway for the 'alternating active sites in a functional dimer' mechanism proposed by the PI and coworkers for this enzyme, specifically by identifying the signal transduction pathway between catalytic centers. Experiments are designed to identify the structural origins of and pathway for dimer-dimer interactions leading to the two different conformations observed by the X-ray studies. In particular, the Pl's group has recently reported kinetics studies indicating that there are two active conformations of the enzyme, one regulated by substrate, the other is not. It is important then to further delineate the structural basis for these two types of conformations and those constructs that can be crystallized will be subjected to high-resolution X-ray methods in an ongoing collaboration with William Furey, Univ. of Pittsburgh, School of Medicine. Goals 1 and 2 will be heavily dependent on the Pl's ability to not only introduce any desired substitution, but also to create tetramers with any desired composition of modified subunits. This enzyme continues to provide an outstanding paradigm not only for thiamin-dependent enzymes, but also for interaction of active centers in homo-tetrameric enzymes.