RUI: Mechanism and Function of Subunit Interactions in Oligomeric Proteins

9986278 Bell摘要：细胞内代谢物水平的精细控制涉及长期适应（包括基因表达控制）和短期调节（包括酶的共价修饰（通常通过磷酸化）或别构调节配体的结合）的组合。变构调节的有效性在寡聚酶中通过亚基相互作用放大，导致调节配体之间或底物结合事件与催化之间的协同相互作用。虽然已经提出了几个变构模型来解释的作用，变构效应可以发挥调节活性，提出的工作将调查的要求，在正常的催化循环的低聚酶，被称为“往复亚基”的亚基相互作用。这项工作的主要目的是了解亚基相互作用在四种重要酶（苹果酸脱氢酶、富马酸酶、3-磷酸甘油酸脱氢酶和谷氨酸脱氢酶）的调节（同向和异向）和活性中所起的作用，这些酶的三维结构都是可用的。这项工作将提供直接的证据支持或反对存在的往复亚基机制与这四种酶。我们还将测试我们的假设，即往复式亚基机制也可能在这些酶的调节中发挥不可或缺的作用，并将测试我们提出的K型或V型效应亚基-亚基相互作用的异向调节剂是否适合这些酶。这项工作将涉及建设一系列的异源寡聚体含有“正常”和修饰位点的组合，以彻底测试的作用，配体诱导的变化，跨亚基接口发挥这些酶的常规活动。此外，与几个晶体学实验室合作，我们将能够获得有关这些酶的活性和调节所涉及的亚基相互作用机制的详细结构信息。

9986278BellAbstract:The exquisite control of the levels of metabolites within the cell involves a combination of both long-term adaptation involving control of gene expression and short-term regulation involving either the covalent modification of an enzyme, frequently by phosphorylation, or the binding of an allosteric regulatory ligand. The effectiveness of allosteric regulation is amplified in oligomeric enzymes by subunit interactions leading to cooperative interactions between regulatory ligands or between substrate binding events and catalysis. While several allosteric models have been presented to explain the roles that allosteric effects can play in the regulation of activity, the work proposed will investigate the requirement for subunit interactions in the normal catalytic cycle of an oligomeric enzyme, referred to as "reciprocating subunits". The major aims of the work are to understand the role that subunit interactions play in the regulation [both homotropic and heterotropic] and activity of four important enzymes, Malate Dehydrogenase, Fumarase, 3-Phosphoglycerate dehydrogenase and Glutamate dehydrogenase, all of whose 3-dimensional structures are available. This work will provide direct evidence for or against the existence of a reciprocating subunit mechanism with these four enzymes. We will also test our hypothesis that reciprocating subunit mechanisms may also play an integral role in regulation of these enzymes, and will test whether our proposed mechanism where heterotropic regulators of either the K-type or the V-type effect subunit-subunit interactions to elicit their effects is appropriate for these enzymes. This work will involve the construction of a series of hetero-oligomers containing combinations of "normal" and modified sites to thoroughly test the roles that ligand induced changes across subunit interfaces play in the regular activity of these enzymes. Furthermore, working in conjunction with several crystallography labs, we will be able to obtain detailed structural information as to the mechanism of the subunit interactions involved in the activity and regulation of these enzymes.