Kinetic and spectroscopic characterization of native and metal-substitutedbeta-lactamase from Aeromonas hydrophila AE036

Two metal ion binding sites are conserved in metallo-beta-lactamase from Ae romonas hydrophila. The ligands of a first zinc ion bound with picomolar di ssociation constant were identified by EXAFS spectroscopy as one Cys, two H is and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(I I) due to ligation of the single conserved cysteine residue. Binding of a s econd metal ion results in non-competitive inhibition which might be explai ned by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed. (C) 2000 Federation of Europea n Biochemical Societies.