ENZYME INTERMEDIATE STRUCTURES BY NMR

通过 NMR 分析酶的中间结构

• 批准号: 6525620
• 负责人: Michael J Smerdon
• 金额: $ 34.37万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525620
• 关键词: active sites; alkyltransferase; bioimaging /biomedical imaging; conformation; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; fluorescence spectrometry; nuclear magnetic resonance spectroscopy; phosphoenolpyruvate; site directed mutagenesis; stable isotope double label; time resolved data

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.

该更新提案要求资金继续我们的学习 酶5-烯醇的结构功能关系-3- 磷酸盐（EPSP）合酶，同时开发一种新方法 一般来说，酶促反应的结构表征 已解决的固态NMR光谱。 该项目将扩展到 包括尿苷二磷酸N-乙酰葡萄糖胺烯醇乙酰葡萄糖转移酶 （UDP-NAG EPT）和3-脱氧-D-Manno-2-二氯磺酸-8-磷酸盐（KD08P） 合酶。 该研究计划旨在确定，使用核 磁共振（NMR）光谱，酶结合的结构 三种烯醇含量转移酶的中间体； EPSP合酶； udp- NAG EPT和KD08P合酶。 EPSP合酶催化凝结 Shikimate-3-磷酸盐和磷酸烯醇丙酮酸，而产品EPSP是一个 芳族氨基酸的生物合成中的关键中间体。 UDP-NAG EPT 是细菌细胞壁生物合成中的关键酶，KD08P合酶是 参与细菌脂多糖生物合成。 具体目标 该更新建议的内容是：（1）执行定向诱变 对EPSP合酶的特定活性位点残基的研究，（2）进行 时间分辨的固态REDOR NMR测量在EPSP合酶上， 测量比最初在GM43215中提出的更长的距离，（3） 将此方法扩展到另外两个烯醇酸酯转移酶，udp-nag EPT和KD08P合酶，如时间允许。 我们相信后果 这些研究特别有趣和令人兴奋，而不仅仅是 扩展我们对EPSP的结构功能关系的理解 合成酶和相关的烯醇酸酯转移酶，但也适用于 开发可以提供详细时间分辨结构的方法学 一般而言，有关酶促反应的信息，甚至是复杂的 诸如LAUE X射线衍射之类的技术难以获得。 这 我们研究的长期目标是与Laue X射线合作 晶体学家，其蛋白质的结构信息全部将 至关重要，并产生酶的分子细节的“移动” 行动。 这可能使抗菌剂在 未来。

项目成果

The H385N mutant of 5-enolpyruvylshikimate-3-phosphate synthase: kinetics, fluorescence, and nuclear magnetic resonance studies.

5-烯醇丙酮莽草酸-3-磷酸合酶的 H385N 突变体：动力学、荧光和核磁共振研究。

• DOI: 10.1006/abbi.1996.0426
• 发表时间: 1996
• 期刊: Archives of biochemistry and biophysics.
• 作者: Shuttleworth,WA;Evans,JN
• 通讯作者: Evans,JN

Time-resolved solid-state REDOR NMR studies of UDP N-acetylglucosamine enolpyruvyl transferase.

UDP N-乙酰氨基葡萄糖烯醇丙酮基转移酶的时间分辨固态 REDOR NMR 研究。

• DOI: 10.1016/0014-5793(95)01338-5
• 发表时间: 1995
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Li,Y;Krekel,F;Ramilo,CA;Amrhein,N;Evans,JN
• 通讯作者: Evans,JN

Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter.

大肠杆菌中 5-烯醇丙酮莽草酸-3-磷酸合酶的过量生产：T7 启动子的使用。

• DOI: 10.1093/protein/5.5.461
• 发表时间: 1992
• 期刊: Protein engineering
• 作者: Shuttleworth,WA;Hough,CD;Bertrand,KP;Evans,JN
• 通讯作者: Evans,JN

The hard-soft acid-base principle in enzymatic catalysis: dual reactivity of phosphoenolpyruvate.

• DOI: 10.1073/pnas.93.10.4612
• 发表时间: 1996-05
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Y. Li;J. N. Evans

Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase.

5-烯醇丙酮莽草酸-3-磷酸合酶的假定活性位点残基的定点诱变。

• DOI: 10.1021/bi9815142
• 发表时间: 1999
• 期刊: Biochemistry.
• 作者: Shuttleworth,WA;Pohl,ME;Helms,GL;Jakeman,DL;Evans,JN
• 通讯作者: Evans,JN