NMR STUDIES OF ENZYME MECHANISMS AND METABOLIC PATHWAYS

这项建议有以下广泛的长远目标：(1) 开发新的核磁共振实验来研究作用中的酶和 解开药物-受体相互作用的结构细节。(2) 了解生物合成酶的详细机制 导致(A)青霉素的几条重要的、复杂的代谢途径， (B)羊毛甾醇和(C)血红素，使用区域特定的高场核磁共振 通过克隆和纯化获得富含13C的底物和纯化的酶 过度表达。(3)基于机理的共价结合研究 在肺气肿疾病中起关键作用的酶抑制剂 (弹性蛋白酶)、艾滋病(HIV-1蛋白酶)和急性间歇性卟啉病(ALA) 合成酶、胆红素原脱氨酶、欧罗三合酶)。(4) 在溶液和固体中的构象和结合方式 抗肿瘤药物长春花碱和紫杉醇对其受体的影响 微管蛋白。(5)细胞内代谢的体内核磁共振研究。 实验方法将结合高场核磁共振波谱在 解决方案(300,500 MHz)和固态(300 MHz)，以及区域和 立体定向合成富含13C的底物和缓蚀剂 直接光谱学和间接光谱学(不合格，不充分，NOESY) (1H/13C)和大口径(20毫米)探针与低温酶学相结合 通过克隆、超量表达和 大于或等于1.0毫摩尔浓度的纯化 这是检测酶结合物种所必需的。 这种方法的重要性，它独特地将有机结合在一起 合成，高场核磁共振，低温(低温)酶学和 分子生物学，即活性部位和药物与受体的相互作用 可以通过结合小分子的同位素进行准确的测绘 分子，最重要的是，生化事件的时间进程 可以通过低温成功地在结构层面上进行监测 核磁共振波谱。

This proposal has the following broad long-term objectives: (1) Development of novel NMR experiments to study enzymes in action and to unravel the structural details of drug-receptor interaction. (2) Understanding the detailed mechanism of the biosynthetic enzymes of several important, complex metabolic pathways leading to (a) penicillin, (b) lanosterol and (c) heme, using high field NMR of regiospecifically 13C enriched substrates and purified enzymes obtained by cloning and overexpression. (3) The study of covalent binding of mechanism-based inhibitors of enzymes which play a key role in the diseases of emphysema (elastase), AIDS (HIV-1 protease) and acute intermittent porphyria (ALA synthase, porphobilinogen deaminase, uro'gen III synthase). (4) The conformations and mode of binding in solution and solid state of the anti-tumor drugs vinblastine (VLB) and taxol to their receptors in tubulin. (5) In vivo NMR studies of intracellular metabolism. The experimental methods will combine high field NMR spectroscopy in solution (300, 500 MHz) and in the solid state (300 MHz) with regio- and stereo-specific synthesis of 13C enriched substrates and inhibitors using direct and indirect (INEPT, INADEQUATE, NOESY) spectroscopy with inverse (1H/13C) and wide bore (20 mm) probes in conjunction with cryoenzymology of the requisite enzymes obtained by cloning, overexpression and purification at greater than or equal to 1.0 millimolar concentrations necessary for the detection of enzyme-bound species. The significance of the approach, which uniquely combines organic synthesis, high field NMR, low temperature (cryo) enzymology and molecular biology, is that active sites and drug-receptor interactions can be accurately mapped via isotopic enrichment of the binding small molecule and, most importantly, the time course of the biochemical events can be successfully monitored at the structural level by low temperature NMR spectroscopy.