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

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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.