Histidine residues of zinc ligands in β-lactamase II

Abstract

The Zn(II)-requiring .beta.-lactamase [EC 3.5.2.6] from Bacillus cereus 569/H/9, which had 2 Zn binding sites, was examined by 270 MHz 1H NMR spectroscopy. Resonances were assigned to 5 histidine residues. Resonances attributed to 3 of the histidine residues in the apoenzyme shift on the addition of 1 equivalent of Zn(II). Although these 3 histidine residues are free to titrate in the apoenzyme, none of them titrates over the pH* range 6.0-9.0 in the mono-Zn enzyme. The ability of the C-2 protons of these three histidine residues to exchange with solvent (2H2O) is markedly decreased on Zn(II) binding. These 3 histidine residues apparently act as Zn ligands at the tighter Zn-binding site. Resonances attributed to a 4th histidine residue shift on addition of further Zn to the mono-Zn enzyme. This histidine residue apparently acts as a Zn(II) ligand at the 2nd Zn-binding site.