STRUCTURE-FUNCTION RELATIONSHIPS OF HUMAN GLUCOKINASE

葡萄糖激酶，肝脏和β细胞中的关键调节酶 糖酵解途径，是己糖激酶家族的一员。 然而，对结构/功能几乎一无所知 这个酶家族的关系。最近发生了一些误会 葡萄糖激酶的突变已经被发现与早期- 起病的非胰岛素依赖型糖尿病梅里炎。这个项目的总体目标是 建议是为了增加我们对分子基础的理解 人肝脏和β细胞葡萄糖激动酶的催化和调节 为了确定葡萄糖激酶突变导致的分子机制 与非胰岛素依赖型糖尿病患者的糖耐量异常有关。它是 建议过表达人肝脏和β细胞葡萄糖激动酶 细菌表达系统，并对它们的动力学和 物理属性。还建议尝试获得x射线 人肝脏和β细胞异构体的晶体结构 这些酶的突变体。将使用经典的化学修饰 研究结构/功能关系。还建议使用 利用定点突变技术鉴定参与基因突变的残基 并确定催化和底物结合的后果 在MODY和其他NIDDM患者中发现葡萄糖激酶突变。在……里面 为了确定人葡萄糖激酶酶突变的后果 肝糖代谢、野生型和突变型葡糖激酶的研究 将在肝癌细胞中表达的酶的数量和类型 将与糖酵解/糖异生和糖原有关 综合。葡萄糖激酶突变起作用的假说 运输活动的主要监管机构也将受到以下方面的考验 野生型和突变型葡萄糖激动酶在att-20ins细胞中的过表达。 突变形式的葡糖激酶也将在转基因小鼠中表达 确定这些突变本身是否会影响胰岛素的分泌 和/或体内肝脏代谢。葡萄糖-6-的结构基础 低KM己糖激酶的磷酸抑制作用将通过 己糖激酶和葡糖激酶嵌合结构的动力学分析 低KM己糖激酶的N-端和C-端的一半。 人葡糖激酶结构/功能关系的研究进展 对人类患者中发现的突变的分析将增强我们的 了解NIDDM的遗传基础并为其他疾病指明方向 基因缺陷，以及对基本机制的新见解 用于调节碳水化合物代谢，最终可能是新的 非胰岛素依赖型糖尿病的治疗方法。

Glucokinase, a key regulatory enzyme in the hepatic and beta-cell glycolytic pathway, is a member of the hexokinase family of enzymes. However, almost nothing is known about the structure/function relationships of this enzyme family. Recently a number of missense mutations in glucokinase have been identified which are linked to early- onset non-insulin dependent diabetes mellitis. The overall goal of this proposal is to increase our understanding of the molecular basis for catalysis and regulation of human liver and beta-cell glucokinases and to define the molecular mechanism by which mutations in glucokinase lead to glucose intolerance in noninsulin dependent diabetes mellitus. It is proposed to overexpress the human liver and beta-cell glucokinases in a bacterial expression system and to characterize their kinetic and physical properties. It is also proposed to attempt to obtain the x-ray crystal structure of the human liver and beta-cell isoforms and of mutants of these enzymes. Classical chemical modification will be used to study structure/function relationships. It is also proposed to use site-directed mutagenesis to identify those residues involved in catalysis and substrate binding and to determine the consequences of mutations in glucokinase found in MODY and other NIDDM patients. In order to determine the consequences of mutations in human glucokinases on hepatic glucose metabolism, wild-type and mutant forms of glucokinase will be expressed in hepatoma cells and the amount and type of enzyme will be related to rates of glycolysis/gluconeogenesis and glycogen synthesis. The hypothesis that glucokinase mutations function as dominant regulators of transporter activity will also be tested by overexpressing wild-type and mutant glucokinases in AtT-20ins cells. Mutated forms of glucokinase will also be expressed in transgenic mice to determine whether these mutations per se affect insulin secretion and/or hepatic metabolism in vivo. The structural basis for glucose-6- phosphate inhibition of low Km hexokinases will be investigated by kinetic analysis of chimeric constructs of hexokinase and glucokinase and of the N-terminal and C-terminal halves of low Km hexokinases. Elucidation of the structure/function relationships of human glucokinase and analysis of mutations found in human patients will enhance our understanding of the genetic basis of NIDDM and point the way to other genetic defects, as well as provide new insights into basic mechanisms for regulation of carbohydrate metabolism and ultimately, perhaps, new modalities for treatment of NIDDM.