Time-dependent structural studies on dinuclear metal ion containing enzymes

含双核金属离子酶的时间依赖性结构研究

• 批准号: 7940321
• 负责人: MICHAEL W CROWDER
• 金额: $ 42.56万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7940321
• 关键词: Active Sites; Address; Amides; Anthrax disease; Antibiotic Resistance; Antibiotics; Arylsulfatases; Bacillus anthracis; Bacterial Infections; Binding; Biochemical Reaction; Catalysis; Cessation of life; Clinic; Clinical; Collection; Coupled; Coupling; Development; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Enzymes; Freezing; Future; Generations; Glean; Gluconolactonase; Goals; Hydrolysis; Ions; Klebsiella pneumonia bacterium; Lactamase; Lactams; Metal Binding Site; Metals; Methodology; Motion; Organism; Penicillin Resistance; Positioning Attribute; Pseudomonas aeruginosa; Reaction; Research; Roentgen Rays; Role; Series; Site; Solutions; Spectrum Analysis; Spin Labels; Stenotrophomonas maltophilia; Structure; Techniques; Time; Transfer RNA; X ray spectroscopy; absorption; base; flexibility; inhibitor/antagonist; metalloenzyme; oxidation; phosphoric diester hydrolase; programs; public health relevance; research study

DESCRIPTION (provided by applicant): This proposal describes studies to probe structural changes in an enzyme during the course of its biochemical reaction. We propose to utilize rapid-freeze quench (RFQ) coupled with several spectroscopic techniques to probe the reaction of metallo-?-lactamase (M?L) L1 from Stenotrophomonas maltophilia as substrate binds and is converted into product. Specifically, ... In Specific aim #1, we propose to explore changes in metal site structure as a function of reaction coordinate, coupling rapid-freeze-quench (RFQ) techniques with conventional spectroscopies, including X-ray absorption spectroscopy (XAS), EPR and electron-nuclear double resonance (ENDOR), in the reaction of L1 with Zn and/or Co at the active site with a ?-lactam substrate. In Specific aim #2, we will examine solution dynamics of a position-conserved flexible loop that covers the metal site during turnover, through CW EPR studies of Co-containing derivatives of L1 incorporating a series of site-directed spin labels (SDSL); these studies will be extended into the time domain using RFQ-EPR to probe larger scale motions of the spin-labeled active site loop in the same Co-L1(SDSL) series. This proposal will offer a new strategy for the characterization of reactions catalyzed by Zn(II)- containing enzymes. More specific to the M?Ls, our long term goal is to identify structural/mechanism details common to all M?Ls, and in the future, we plan to apply the approaches, developed in this proposal, to other distinct M?Ls. The methodologies developed herein will easily lend themselves to the study of other metalloenzymes, regardless of the identity, oxidation level, or spin state of the metal. PUBLIC HEALTH RELEVANCE: The emergence of antibiotic resistance in the clinic has resulted in a biomedical crisis in which bacterial infections, once treated with inexpensive antibiotics, are now untreatable and cause a large number of deaths annually in the US and throughout the world. This proposal describes experiments to probe a metallo-?-lactamase, which causes resistance to penicillin and other ?-lactam containing antibiotics in the clinic, as it proceeds during catalysis. The information gleaned in these studies can be used to guide in the future development of M?L inhibitors.

描述（由申请人提供）：该提案描述了在其生化反应过程中探测酶结构变化的研究。我们建议利用快速冻结（RFQ）以及几种光谱技术来探测嗜嗜性嗜嗜性嗜性嗜性元素的金属 - ？？ - lactamase（m？l）L1作为底物结合，并将其转化为产物。 Specifically, ... In Specific aim #1, we propose to explore changes in metal site structure as a function of reaction coordinate, coupling rapid-freeze-quench (RFQ) techniques with conventional spectroscopies, including X-ray absorption spectroscopy (XAS), EPR and electron-nuclear double resonance (ENDOR), in the reaction of L1 with Zn and/or Co at the active site with a ?-lactam substrate.在特定的目标＃2中，我们将通过CW EPR研究在营业额期间覆盖金属​​位置的位置保存的柔性环的解决方案动力学，该循环涵盖了L1的共同辅助衍生物，该衍生物结合了一系列位置定向的自旋标记（SDSL）；这些研究将使用RFQ-EPR扩展到时域中，以探测同一CO-L1（SDSL）系列中自旋标记的活性位点环的较大规模运动。该提案将为含有酶的Zn（II）催化的反应的表征提供新的策略。我们的长期目标是更具体的是，我们的长期目标是确定所有M级常见的结构/机制细节，并且将来我们计划将本提案中开发的方法应用于其他不同的M？ls。本文开发的方法将很容易地研究其他金属酶的研究，而不管金属的身份，氧化水平或旋转状态如何。 公共卫生相关性：诊所中抗生素耐药性的出现导致了一场生物医学危机，在这种危机中，曾经接受过廉价抗生素治疗的细菌感染现在无法治疗，并每年在美国和全世界造成大量死亡。该建议描述了探测金属 - ？ - 乳糖酶的实验，这会导致对青霉素和其他含有抗生素的抗生素的耐药性，因为它在催化过程中进行了抗生素。这些研究收集的信息可用于指导M？L抑制剂的未来发展。

项目成果

New β-phospholactam as a carbapenem transition state analog: Synthesis of a broad-spectrum inhibitor of metallo-β-lactamases.

• DOI: 10.1016/j.bmcl.2013.08.098
• 发表时间: 2013-11-01
• 期刊: BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
• 影响因子: 2.7
• 作者: Yang, Ke-Wu;Feng, Lei;Yang, Shao-Kang;Aitha, Mahesh;LaCuran, Alecander E.;Oelschlaeger, Peter;Crowder, Michael W.
• 通讯作者: Crowder, Michael W.