Time-dependent structural studies on dinuclear metal ion containing enzymes

描述(由申请人提供)：这项建议描述了探索酶在其生化反应过程中的结构变化的研究。我们建议利用快速冷冻猝灭技术结合多种光谱技术来探索嗜麦芽窄食单胞菌的金属内酰胺酶(M？L)L1作为底物结合并转化为产物的反应。具体地说，..。在特定的目标#1，我们建议探索金属中心结构的变化作为反应坐标的函数，将快速冷冻猝灭(RFQ)技术与常规光谱技术相结合，包括X射线吸收光谱(XAS)、电子顺磁共振(EPR)和电子-核双共振(Endor)，在L1与活性中心的锌和/或钴与β-内酰胺底物的反应中。在特定的目标#2中，我们将通过包含一系列位置定向自旋标记(SDSL)的L1的含钴衍生物的连续波EPR研究，来研究在周转过程中覆盖金属位置的位置守恒的柔性环的溶液动力学；这些研究将被扩展到时间域，使用RFQ-EPR来探索同一Co-L1(SDSL)系列中自旋标记的活性中心环的更大规模的运动。这一建议将为表征含锌(II)酶催化的反应提供新的策略。更具体地说，我们的长期目标是确定所有M？LS共同的结构/机制细节，在未来，我们计划将本提案中开发的方法应用于其他不同的M？LS。无论金属的特性、氧化程度或自旋状态如何，本文开发的方法都将很容易地用于其他金属酶的研究。 与公共卫生相关：临床上出现的抗生素耐药性已经导致了一场生物医学危机，一旦用廉价的抗生素治疗，细菌感染现在就无法治愈，每年在美国和世界各地导致大量死亡。这项建议描述了一种金属内酰胺酶的实验，这种酶在催化过程中会导致临床上对青霉素和其他含有抗生素的β-内酰胺类抗生素产生耐药性。这些研究收集的信息可以用来指导M？L抑制剂的未来开发。

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.