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）：本提案描述了在酶的生化反应过程中探测其结构变化的研究。我们建议利用快速冷冻淬灭（rapid-freezquenchment，简称FQQ）结合几种光谱技术来探测金属-？-内酰胺酶（M？L）来自嗜麦芽窄食单胞菌的L1作为底物结合并转化为产物。具体来说，在具体目标#1中，我们提出探索金属位点结构的变化作为反应坐标的函数，将快速冷冻淬灭（Rapid Freeze-Quench，缩写为REQ）技术与常规光谱学（包括X射线吸收光谱（XAS）、EPR和电子-核双共振（ENDOR））相结合，在L1与Zn和/或Co在活性位点与？内酰胺底物。在具体目标#2中，我们将通过包含一系列定点自旋标记（SDSL）的L1含Co衍生物的CW EPR研究，研究在周转期间覆盖金属位点的位置守恒柔性环的溶液动力学;这些研究将使用RFQ-EPR扩展到时域，以探测相同Co-L1（SDSL）系列中自旋标记活性位点环的更大尺度运动。这一建议将提供一个新的策略，由含锌（II）的酶催化的反应的表征。具体到M？LS，我们的长期目标是确定结构/机制的细节共同所有M？LS，在未来，我们计划应用的方法，在这个建议中开发的，其他不同的M？LS.本文开发的方法将很容易地适用于其他金属酶的研究，无论身份，氧化水平，或自旋状态的金属。 公共卫生相关性：抗生素耐药性在临床上的出现导致了生物医学危机，其中细菌感染，一旦用廉价的抗生素治疗，现在是无法治疗的，每年在美国和世界各地造成大量死亡。该提案描述了探测金属-？-内酰胺酶，导致对青霉素和其他？在临床上，它是含有抗生素的内酰胺，因为它在催化期间进行。在这些研究中收集的信息可以用来指导在未来的发展M？L抑制剂。

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.