Structure and Mechanism of Yeast Pyruvate Decarboxylase

描述(申请人提供)：这项研究的总体目标是大大提高我们对结构-功能关系的理解，包括酵母丙酮酸脱羧酶的机制和调节。在酒精发酵的倒数第二步，这种酶需要硫胺素二磷酸(维生素B1辅酶)和脱羧丙酮酸。要求为下一阶段继续研究该酶提供资金，主要目标如下：(1)利用各种光谱方法进一步研究催化残留物的作用。通过使用中间分配和快速猝灭方法直接评估所有关键的二磷酸硫胺素结合中间体的浓度来确定所有活性中心变体以及野生型酶中的限速步骤；(2)由PI和其同事为该酶提出的旨在确定“功能二聚体中交替活性位点”机制的结构起源和途径的实验，特别是通过确定催化中心之间的信号转导途径。实验旨在确定二聚体-二聚体相互作用的结构来源和途径，从而导致X射线研究观察到的两种不同构象。特别是，Pl的小组最近报道了动力学研究，表明该酶有两种活性构象，一种受底物调节，另一种不受底物调节。因此，重要的是要进一步描绘这两种构象的结构基础，那些可以结晶的构象将在与威廉·富雷大学的持续合作下接受高分辨率X射线方法。匹兹堡医学院。目标1和目标2将在很大程度上依赖于PL的能力，不仅引入任何所需的取代，而且创建具有任何所需的修饰亚基组成的四聚体。这种酶不仅为硫胺素依赖的酶，而且也为同源四聚体酶中活性中心的相互作用提供了一个出色的范例。

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.