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

脆弱类杆菌B类含锌金属β-内酰胺酶的催化作用的结构基础将通过使用低温酶学、电子核双共振(Endor)光谱和计算机辅助分子模拟的实验方法来确定。CCRA金属-β-内酰胺酶的反应中间产物将使用低温动力学方法、液体有机-水冷溶剂混合物和合成自旋标记的青霉素或头孢菌素底物作为光谱探针进行动力学测定。反应中间体将通过冷冻猝灭溶液的方式稳定下来，以便收集Endor数据，其方式类似于用于冷冻结晶学的蛋白质晶体的冷冻猝灭。野生型重组酶的活性中心残基将通过氢/氚交换或氟-色氨酸的生物合成掺入来进行Endor光谱的同位素富集化。Endor将测量活性中心残基和底物上的同位素标记核的主要超精细耦合成分，以确定它们的穿透空间(偶极)超精细耦合成分，并估计相应的电子-核距离。通过计算机辅助分子图形分析，将偶极电子-核距离作为约束条件来指定活性中心结构和底物构象。为了确定活性中心锌离子和金属配位水的结构作用，将使用顺磁性的锌离子/钴离子和锌离子/镉杂化酶。Co2+中心将不仅作为通过使用H217O确定内部球体配位水的存在的顺磁位置，而且还将作为化学探针来识别管理kcat和kcat/km的金属连接电离。通过测定CCRA酶反应中间体的底物构象、活性中心结构和金属离子配位几何构型，并与稳态动力学参数的pH曲线相关联来指定金属连接的电离，将确定酶催化反应的机理，并指定锌离子和金属配位溶剂的化学和结构作用。Endor方法已经证明，对于氮氧基自旋标记和VO2+，电子-核距离的估计不确定度在3-11A范围内小于5%。酶活性部位的催化活性结构将通过受Endor确定的电子-核距离限制的分子图形分析来指定。这些结合的结果将使我们更详细地了解活性中心双核金属离子簇在CCRAβ-内酰胺酶催化的β-内酰胺类抗生素水解中的结构和化学作用，以及双核金属-β-内酰胺酶与其丝氨酸水解酶的作用机制是如何不同的。

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.