Structural Basis of Action of the CcrA Metallo-beta-lactamase

CcrA 金属-β-内酰胺酶作用的结构基础

• 批准号: 0092524
• 负责人: Marvin Makinen
• 金额: $ 30万
• 依托单位: University of Chicago
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0092524&HistoricalAwards=false
• 关键词: Structural; Basis; Action; CcrA; Metallo

Makinene, Marvin W.MCB-0092524The structural basis of catalytic action of the class B, Zn2+-containing, CcrA metallo-beta-lactamase of Bacteroides fragilis will be assigned through an experimental approach employing cryoenzymology, electron nuclear double resonance (ENDOR) spectroscopy, and computer-assisted molecular modeling. Reaction intermediates of the CcrA metallo-beta-lactamase will be determined kinetically with use of cryokinetic methods, fluid organic-aqueous cryosolvent mixtures, and synthetic spin-labeled penicillin or cephalosporin substrates as spectroscopic probes. Reaction intermediates will be stabilized for ENDOR data collection by freeze-quenching of the solution in a manner similar to that employed in freeze-quenching of protein crystals for cryo-crystallography. Active site residues of wild type recombinant enzyme will be enriched with isotopes by hydrogen/deuterium exchange or biosynthetic incorporation of fluoro-tryptophan for ENDOR spectroscopy. The principal hyperfine coupling components of isotopically labeled nuclei in active site residues and on the substrate will be measured by ENDOR to determine their through-space (dipolar) hyperfine coupling components and to estimate corresponding electron-nucleus distances. The dipolar electron-nucleus distances will be used as constraints to assign active site structure and substrate conformation by computer assisted molecular graphics analysis. To assign the structural roles of the active site Zn2+ ions and of metal-coordinated water, paramagnetic Zn2+/Co2+ and Zn2+/Cd2+-hybrid enzymes will be used. The Co2+-center will serve not only as the paramagnetic site for determining the presence of inner-sphere coordinated water through use of H217O but also as a chemical probe to identify metal-linked ionizations governing kcat and kcat/KM. By determining substrate conformation, active site structure, and metal ion coordination geometry in a reaction intermediate of the CcrA enzyme, correlated with the pH profiles of steady-state kinetic parameters to assign metal-linked ionizations, the mechanism of the enzyme catalyzed reaction will be determined and the chemical and structural roles of the Zn2+ ions and metal-coordinated solvent will be assigned.The ENDOR methods have been shown to yield estimates of electron-nucleus distances with less than 5% uncertainty in the 3-11 A range for nitroxyl spin-labels and in the 3-8A range for VO2+. The catalytically competent structure of the active site of the enzyme will be assigned through molecular graphics analysis constrained by ENDOR determined electron-nucleus distances. The combined results will lead to a detailed understanding of the structural and chemical roles of the active site binuclear metal ion cluster in the hydrolysis of beta-lactam antibiotics catalyzed by the CcrA beta-lactamase and how the mechanism of the bi-nuclear metallo-beta-lactamases differs from that of their serine hydrolase counterparts.

Makinene，Marvin W. MC B-0092524脆弱拟杆菌的B类含Zn 2+的CcrA金属β-内酰胺酶催化作用的结构基础将通过采用冷冻酶学、电子核双共振（ENDOR）光谱和计算机辅助分子建模的实验方法进行归属。CcrA金属-β-内酰胺酶的反应中间体将使用冷冻动力学方法、流体有机-水性冷冻溶剂混合物和合成自旋标记青霉素或头孢菌素底物作为光谱探针进行动力学测定。 通过以类似于用于冷冻结晶学的蛋白质晶体的冷冻淬灭中所采用的方式对溶液进行冷冻淬灭，稳定反应中间体，以用于ENDOR数据收集。 野生型重组酶的活性位点残基将通过氢/氘交换或氟色氨酸的生物合成掺入富集同位素，用于ENDOR光谱法。 将通过ENDOR测量活性位点残基中和基底上的同位素标记核的主要超精细耦合组分，以确定其通过空间（偶极）的超精细耦合组分，并估计相应的电子-核距离。偶极电子-核距离将被用作限制条件，通过计算机辅助分子图形分析来指定活性位点结构和底物构象。为了分配活性位点Zn 2+离子和金属配位水的结构作用，将使用顺磁性Zn 2 +/Co 2+和Zn 2 +/Cd 2 +-杂合酶。的Co 2+中心将不仅作为顺磁性网站，通过使用H217 O确定内球配位水的存在，但也作为化学探针，以确定金属连接的电离管理kcat和kcat/KM。通过确定CcrA酶的反应中间体中的底物构象、活性位点结构和金属离子配位几何形状，与稳态动力学参数的pH曲线相关联以分配金属连接的电离，酶催化反应的机理将被确定，并且Zn 2+离子和金属离子的化学和结构作用将被确定。ENDOR方法已经显示出产生电子-核距离的估计，对于硝酰基自旋标记物在3-11 A范围内和对于VO 2+在3- 8 A范围内具有小于5%的不确定性。 酶的活性位点的催化活性结构将通过由ENDOR确定的电子-核距离约束的分子图形分析来分配。结合的结果将导致详细了解的结构和化学作用的活性位点的双核金属离子簇的β-内酰胺抗生素的水解催化的CcrA β-内酰胺酶和如何的双核金属β-内酰胺酶的机制不同于他们的丝氨酸水解酶对应物。