IDENTIFICATION OF THE HISTIDINE LIGANDS TO THE BINUCLEAR METAL CENTER OF PHOSPHOTRIESTERASE BY SITE-DIRECTED MUTAGENESIS

In order to identify which of the seven histidines in phosphotriesterase participate at the active site/binuclear metal center of the enzyme, site-directed mutagenesis has been employed to change, individually, each of the seven histidine residues to asparagine. In addition, the gene for the wild-type enzyme has been subcloned without its leader sequence behind a modified ribosomal binding site, leading to a 5-fold increase in protein expression. The seven mutants, H55N, H57N, H123N, H201N, H230N, H254N, and H257N, exhibit varying degrees of activity compared to the wild-type enzyme. The H123N and H257N mutants are as active as the wild-type enzyme, but all of the other mutant enzymes have 10% or less activity. The metal content of the cobalt-purified mutant enzymes has been determined to be less than that of the wild-type enzyme in all cases. Each of the mutant enzymes has been converted to apoenzyme and reconstituted with 2 equiv of zinc(II), cadmium(II), or cobalt(II). The kinetic parameters, V(max) and V/K(m), and apparent pK(a)'s have been determined for each of the reconstituted enzyme derivatives. In almost all cases, the apparent pK(a)'s have shifted toward higher values. The pH-rate profiles for some of the reconstituted mutant enzymes are significantly different from those for the wild-type enzyme, indicating that other groups may become involved in the reaction mechanism upon mutation of the histidine residue to asparagine. His-123 is the only histidine residue that appears to have no involvement in the catalytic activity of phosphotriesterase. The other six histidine residues appear to be involved in some capacity, either as metal ligands or as the catalytic base or proton-shuttle group. A working model has been proposed that incorporates six of the histidines into the active site/metal centers of phosphotriesterase. The two closely spaced pairs, His-55/His-57 and His-254/His-257, are proposed to act as bidentate ligands, with one pair liganding to the primary catalytic metal and the other pair liganding to the secondary metal, which provides structural and catalytic support. His-230 is proposed to act as the bridging ligand between the two metal ions, and His-201 is proposed to be the catalytic base or proton-shuttle group.