Structural Determinants of Glycogen Initiation

描述(由申请人提供)：本提案的总体目标是研究糖原蛋白的结构和功能特性，这些特性导致其独特的自糖基化活性，从而启动糖原的合成。糖原是葡萄糖的重要储备物，糖原合成的启动和合成机制从酵母到人类都是保守的。这一过程的异常调节与各种糖原储存疾病和II型糖尿病有关。这一建议的长期目标是为糖原蛋白催化的自动糖基化反应提供结构背景，并提供关于催化这一不寻常反应的机制的新信息。虽然我们最近的结构测定并没有证明一种特定的机制优于另一种机制，但它确实为我们提供了一个新的起点，从化学和动力学上探索催化的特异性，这是糖原合成的起始步骤。这项工作将为全面了解糖原蛋白的功能和结构性质提供基础，并将有助于对糖原颗粒的合成以及糖原蛋白在这一重要细胞过程中所起的作用提供新的见解。在这项工作的执行过程中，我们将1)确定全长糖原的三维结构，以了解蛋白质可能如何与糖原合成酶相互作用。2)通过获得酶与底物和受体分子的络合物的结构，研究底物和受体结合的结构决定因素。3)评估亚单位相互作用在糖基转移酶反应的初始和后续阶段中所起的作用。4)测试糖原活性部位的特定残基在催化过程中的作用以及对底物和受体分子的识别作用。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to examine the structural and functional properties of glycogenin that lead to its unique self-glucosylation activity that primes the synthesis of glycogen. Glycogen is an important storage reserve of glucose and the mechanism by which the synthesis of glycogen is initiated and synthesized is conserved from yeast to humans. Aberrant regulation of this process is associated with various glycogen-storage diseases and Type II Diabetes. The long-range goal of this proposal is to provide both a structural context for the auto-glucosylation reaction catalyzed by glycogenin and new information with regard to the mechanism by which this unusual reaction is catalyzed. While our recent structure determination has not proven one particular mechanism over another, it does provide us with a new starting point from which to probe the specifics of catalysis, both chemically and kinetically, that underlie the initiation step of glycogen synthesis. The work described in this proposal will provide a foundation for a complete understanding of the functional and structural properties of glycogenin and will contribute new insight into the synthesis of a glycogen particle and the role that glycogenin plays in this important cellular process. During the performance of this work we will 1) Determine the three-dimensional structure of full-length glycogenin, to understand how the protein might interact with glycogen synthase. 2) Examine the structural determinants of substrate and acceptor binding by obtaining structures of the enzyme in complex with both substrate and acceptor molecules. 3) Assess the role that subunit interactions play in both the initial and subsequent phases of the glycosyltransferase reaction. 4) Test the proposed roles for specific residues in the active site of glycogenin during catalysis and for the recognition of substrate and acceptor molecules.