MOLECULAR MECHANISM OF HMG COA REDUCTASE

我们的实验室正在进行多方面的研究项目， 了解酶3-羟基-3- 甲基戊二酰辅酶A（HMG-CoA）还原酶， 生物物理学和X射线晶体学方法。从2.8A结构 的酶和一些二元和三元 配合物与底物，我们已经概述了催化机制， 我们认为适用于这类酶的所有成员。我们现在 建议研究机械建议， 晶体学和动力学的措施，与目标的捕获 催化反应中结构上重要的中间体，和 将把这些研究扩展到一些人的HMG-CoA还原酶， 不同的物种我们还将开始调查 可逆酶调节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 活性位点的物种特异性差异的调查 来自细菌、古细菌和真核生物的HMG-CoA还原酶， 导致这种酶的物种选择性抑制剂的发展。 本提案的具体目标是：1）完成 酶-底物复合物的晶体学研究 假单胞菌HMG-CoA还原酶的分子机制。（二） 使用稳态和快速反应动力学与可能的自杀 抑制剂，以确定和表征在 假单胞菌HMG-CoA还原酶反应，其可以通过X- 射线晶体学技术，并将这些研究扩展到 硫化叶菌和叙利亚仓鼠酶。3)调查的依据 用X射线晶体学控制HMG-CoA还原酶的磷酸化 一种工程假单胞菌酶的研究。4)表达，净化， 表征并结晶来自于该酶的代表性HMG-CoA还原酶。 I类和II类酶，以检查观察到的 它们的生物学和生物化学特性的差异被表达出来， in their其structures结构. 5)利用晶体结构 酶来识别差异， 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.