Role of aminoacidic residues inside active sites of metalloproteins

The role of the residues inside the active cavity of metalloproteins can be investigated by using the metal ion itself as a spectroscopic probe. For example in carbonic anhydrase the cobalt(I1) substituted enzyme has allowed us to understand the role of a free histidine hanging in the cavity in lowering the pK of the coordinated water. Substitution of zinc with cobalt(fi) in superoxide dismutase has allowed us to check the spatial arrangement of metal ligands and the mode of inhibitor binding when important residues like Arg 143 in the human enzyme are substituted through site directed mutagenesis. The cobalt(I1) substituted derivative of carboxypeptidase A has been used to show that molecules with carboxylate groups bind at Arg 145. Afterwards ternary complexes with metal ligands like azide can be obtained. It is proposed that the binding of a carboxylate function to the guanidinium group of Arg 145 is the key step in modulating the opening of the cavity and the access to the metal of substrates and inhibitors.