ENZYME INTERMEDIATE STRUCTURES BY NMR

这份续签提案要求提供资金继续我们对他的研究 5-烯醇丙酮基莽草酸-3-烯醇酯酶的构效关系 磷酸(EPSP)合成酶，同时进一步开发一种新的方法 酶反应的结构表征一般按时间- 可分辨的固态核磁共振波谱。该项目将扩大到 包括尿苷二磷酸N-乙酰氨基葡萄糖烯醇式丙酮基转移酶 (UDP-NAG EPT)和3-脱氧-D-甘露-2-辛基-8-磷酸(KD08P) 合成酶。这项研究计划旨在利用核能来确定 核磁共振(核磁共振)谱，结合酶的结构 三种烯醇式丙酮转移酶的中间体；EPSP合成酶；UDP- NAG EPT和KD08P合成酶。EPSP合酶催化缩合 莽草酸-3-磷酸和磷酸烯醇式丙酮酸，产物EPSP是一种 芳香族氨基酸生物合成的关键中间体。UDP-NAG EPT 是细菌细胞壁生物合成的关键酶，KD08P合成酶是 参与细菌脂多糖的生物合成。具体目标 这一更新建议的目的是：(1)进行定点突变 EPSP合成酶特定活性部位残基的研究，(2)开展 EPSP合成酶的时间分辨固体红外线核磁共振测量， 测量的距离比GM43215中最初建议的更长，以及(3) 将这种方法扩展到另外两种烯醇式丙酮酸转移酶，UDP-NAG EPT和KD08P合酶，只要时间允许。我们相信，这些后果 这些研究中特别有趣和令人兴奋的，不仅仅是 扩大对EPSP结构-功能关系的认识 合成酶和相关的烯醇式丙酮基转移酶，也用于 开发能够提供详细的时间解析结构的方法 关于酶反应的一般信息，甚至是复杂的 像劳厄X射线衍射这样的技术很难获得。这个 我们研究的长期目标是与劳厄X射线合作 结晶学家，他将蛋白质的结构信息作为整体 至关重要，并产生一种酶的分子细节的“移动” 行动。这可能使抗菌剂的合理使用成为可能。 未来。

This renewal proposal requests funds to continue our studies ont he structure-function relationships for the enzyme 5-enolpyruvylshikimate-3- phosphate (EPSP) synthase, while developing further a new method for the structural characterization of enzymatic reactions in general by time- resolved solid-state NMR spectroscopy. The project will be extended to include uridine diphosphate N-acetyl-glucosamine enolpyruvyl transferase (UDP-NAG EPT) and 3-deoxy-D-manno-2-octulosonate-8-phosphate (KD08P) synthase. The research program is designed to determine, using nuclear magnetic resonance (NMR) spectroscopy, the structure of the enzyme-bound intermediates of three enolpyruvyl transfer enzymes; EPSP synthase; UDP- NAG EPT and KD08P synthase. EPSP synthase catalyzes the condensation of shikimate-3-phosphate and phosphoenolpyruvate, and the product, EPSP, is a key intermediate in the biosynthesis of aromatic amino acids. UDP-NAG EPT is a key enzyme in bacterial cell wall biosynthesis, and KD08P synthase is involved in bacterial lipopolysaccharide biosynthesis. The specific aims of this renewal proposal are to: (1) carry out site-directed mutagenesis studies on specific active site residues of EPSP synthase, (2) carry out time-resolved solid-state REDOR NMR measurements on EPSP synthase, measuring longer distances than was originally proposed in GM43215, and (3) extend this approach to two other enolpyruvyl transferase enzymes, UDP-NAG EPT and KD08P synthase, as time permits. We believe that the consequences of these studies are particularly interesting and exciting, not just for extending our understanding of the structure-function relationship of EPSP synthase and related enolpyruvyl transferase enzymes, but also for developing methodologies that can provide detailed time-resolved structural information on enzymatic reactions in general, which even sophisticated techniques like Laue X-ray diffraction have difficulty obtaining. The long-term goal of our research is to collaborate with a Laue X-ray crystallographer, whose structural information of the protein as whole will be crucial, and generate a "move" of the molecular details of an enzyme in action. This might enable the rational of antibacterial agents in the future.