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, Vmax and V/Km, and apparent pKa's have been determined for each of the reconstituted enzyme derivatives. In almost all cases, the apparent pKa'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.(ABSTRACT TRUNCATED AT 250 WORDS)
Kuo
J M
JM
Department of Chemistry, Texas A&M University, College Station 77843.
Raushel
F M
FM
D043303:Aryldialkylphosphatase; D002104:Cadmium / Q000378:metabolism; D002942:Circular Dichroism; D003035:Cobalt / Q000378:metabolism; D004789:Enzyme Activation; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008670:Metals / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D008024:Ligands; D008670:Metals; D011994:Recombinant Proteins; D002104:Cadmium; D003035:Cobalt; D006639:Histidine; D004950:Esterases; D043303:Aryldialkylphosphatase; D015032:Zinc