MOLECULAR MECHANISM OF HMG-COA REDUCTASE

我们的实验室正在进行一个了解分子的项目。 3-羟基-3-甲基戊二酰辅酶A的作用机理 用X射线结晶学方法测定还原酶的含量。HMG-CoA还原酶催化 HMG-CoA与甲氧丙戊酸的相互转化是一种根部反应 导致类异戊二烯类脂合成的途径包括 胆固醇及其衍生物。在哺乳动物中，这种反应是第一次 致力于胆固醇的生物合成，因此HMG-CoA还原酶 被认为是#年控制胆固醇产生的主要目标 活着。我们已经解决了假单胞菌的2.8埃结构 哺乳动物的催化和结构模型--Mevalonii酶 酵素。这种细菌HMG-CoA还原酶的结构揭示了一种 紧密结合的二聚体具有明确的活性部位裂隙位于 二聚体界面。初步研究表明，结晶酶- 可以产生底物复合体，其指示参与 底物的结合和酶的催化活性。 在这项研究中，我们建议扩展我们对HMG-CoA还原酶的研究 以提高天然酶和酶底物的分辨率 络合物，开始分子的分子解剖与位置- 定向诱变和利用这种细菌酶结构作为 研究哺乳动物HMG-CoA还原酶及其相互作用的模型 服用降胆固醇药物。我们的具体目标是：1)扩大 当前结构到2.4埃分辨率，2)产生二进制 该酶的所有底物与稳定的非产酶的络合物 三元络合物，3)收集关于这些络合物的高分辨率数据 并改进它们的结构，以便研究 分子机制；4)用结晶学方法研究 这些建议的关键残基中含有突变体的酶 复合体。为了研究哺乳动物系统，我们将对 哺乳动物HMG-CoA还原酶的结构 细菌酶和6)研究降胆固醇药物的结合， 首先是对P.mevalonii还原酶和能量技术的研究 最小化，以模型哺乳动物的结构。最后，我们将延长 这些结构研究试图使催化剂结晶 叙利亚仓鼠HMG-CoA还原酶的结构域，8)通过研究 解决这一C-末端区域结构的可能性 分子的核磁共振研究和开始结晶试验。 其他与HMG-CoA代谢有关的酶、HMG-CoA合成酶和HMG-CoA 裂解酶。

Our laboratory is pursuing a project to understand the molecular mechanism of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by X-ray crystallographic methods. HMG-CoA reductase catalyzes the interconversion of HMG-CoA and mevalonate, a reaction at the root of the pathway leading to synthesis of isoprenoid lipids, including cholesterol and its derivatives. In mammals this reaction is the first committed step in cholesterol biosynthesis, and so HMG-CoA reductase is considered a primary target for the control of cholesterol production in vivo. We have solved the 2.8 angstroms structure of the Pseudomonas mevalonii enzyme, a catalytic and structural model for the mammalian enzyme. The structure of this bacterial HMG-CoA reductase reveals a tightly bound dimer with a well defined active site cleft located at the dimer interface. Preliminary studies have shown that crystalline enzyme- substrate complexes can be produced which indicate residues involved in the binding of the substrates and the catalytic activity of the enzyme. In this research we propose to extend our studies of HMG-CoA reductase to higher resolution for both the native enzyme and the enzyme-substrate complexes, to begin a molecular dissection of the molecule with site- directed mutagenesis and to use this bacterial enzyme structure as a model to investigate the mammalian HMG-CoA reductase and its interactions with anticholesterol drugs. Our specific aims are 1) to extend the current structure to 2.4 angstroms resolution, 2) to produce binary complexes of all the substrates of this enzyme and stable nonproductive ternary complexes, 3) to collect high resolution data on these complexes and refine their structures in order to investigate the details of the molecular mechanism and 4) to study by crystallographic methods the enzymes containing mutants in critical residues suggested by these complexes. In order to study the mammalian system we will 5) model the structure of the mammalian HMG-CoA reductase on the structure of the bacterial enzyme and 6) investigate the binding of anticholesterol drugs, first to the P. mevalonii reductase and the, using techniques of energy minimization, to the model mammalian structure. Finally we will extend these structural studies by 7) attempting to crystallize the catalytic domain of the Syrian hamster HMG-CoA reductase, 8) by investigating the possibility of solving the structure of the C-terminal region of this molecule by an NMR study and 9) by beginning crystallization trials on other enzymes related to HMG-CoA metabolism, HMG-CoA synthase and HMG-CoA lyase.