MOLECULAR MECHANISM OF HMG COA REDUCTASE

我们实验室正在进行多方面的研究项目，以 了解3-羟基-3-羟基酶的分子机制 甲基戊二酰辅酶A(HMG-CoA)还原酶 生物物理和X射线结晶学方法。从2.8A的结构 对梅瓦隆假单胞菌及若干二元和三元酶的研究 与底物的络合物，我们已经概述了一种催化机理 我们认为适用于这类酶的所有成员。我们现在 建议结合以下几个方面来研究机械性提案 以捕获为目标的结晶学和动力学措施 催化反应中具有重要结构意义的中间体，以及 将把这些研究扩展到一些HMG-CoA还原酶 不同的物种。我们还将开始调查结构 可逆性调节HMG-CoA还原酶活性的基础 哺乳动物酶中的磷酸化，同时试图 使哺乳动物的一种还原酶结晶。最后，我们打算开始 黄曲霉活性部位的种属差异研究 细菌、弓形虫和真核生物的HMG-CoA还原酶，可能 导致了该酶的物种选择性抑制剂的开发。 这项建议的具体目的是：1)完成 酶-底物复合体的结晶学研究 梅瓦隆假单胞菌HMG-CoA还原酶的分子机制。2) 使用稳态和快速反应动力学，有可能自杀 用于识别和表征在生产过程中产生的中间体的抑制剂 X-射线能检测假单胞菌HMG-CoA还原酶反应 射线结晶学技术，并将这些研究扩展到 Sulfolobus和叙利亚仓鼠酵素。3)调查基础 X射线结晶学研究HMG-CoA还原酶的磷酸化调控 一种工程假单胞菌酶的研究。4)表达、净化、 具有代表性的HMG-CoA还原酶的鉴定和结晶 为了检查观察到的I类和II类酶 它们在生物学和生化特性上的差异被表达出来 在他们的结构中。5)使用这些材料的晶体结构 酶来识别可被利用来设计的差异 HMG-CoA还原酶的种类/特异性抑制剂。

Our laboratory is pursuing a multi-faceted research projects to understand the molecular mechanism of the enzyme 3-hydroxyl-3- methylglutaryl co-enzyme A (HMG-CoA) reductase by a combination of biophysical and X-ray crystallographic methods. From the 2.8A structure of the Pseudomonas mevalonii enzyme and a number of binary and ternary complexes with substrates, we have outlined a catalytic mechanism which we believe applies to all members of this class of enzymes. We now propose to investigate the mechanistic proposal with a combination of crystallographic and kinetic measures, with the goal of trapping structurally significant intermediates in the catalytic reaction, and will extend these studies to the HMG-CoA reductases of a number of different species. We will also begin to investigate the structural basis of the modulation of activity in HMG-CoA reductase by reversible phosphorylation in the mammalian enzymes, while attempting to crystallize one of the mammalian reductases. Finally, we intend to begin investigations of species specific differences in the active sites of HMG-CoA reductases from bacteria, archeabacter and eukaryotes which may lead to the development of species-selective inhibitors for this enzyme. The specific aims of this proposal are to: 1) Complete the crystallographic studies of the enzyme-substrate complexes involved in the molecular mechanism of Pseudomonas mevalonii HMG-CoA reductase. 2) Use steady-state and fast reaction kinetics with possible suicide inhibitors to identify and characterize intermediates produced during the Pseudomonas HMG-CoA reductase reaction which can be examined by X- ray crystallographic techniques, and to extend these studies to the Sulfolobus and Syrian hamster enzymes. 3) Investigate the basis of phosphorylation control of HMG-CoA reductase with X-ray crystallographic studies of an engineered Pseudomonas enzyme. 4) Express, purify, characterize and crystallize representative HMG-CoA reductases from the Class I and Class II enzymes in order to examine how the observed differences in their biological and biochemical properties are expressed in their structures. 5) Use the crystallographic structures for these enzymes to identify differences that could be exploited to design species/specific inhibitors for HMG-CoA reductase.