Bimetallic binding motifs in organophosphorus hydrolase are important for catalysis and structural organization.
Organophosphorus hydrolase is a broad spectrum phosphoric acid hydrolase (EC 3.1.8.1) which appears to contain a binuclear metal center with two metals interactively involved in catalysis and/or structural functions. Site-directed mutagenesis has been employed to evaluate the participation of the various histidine and cysteine residues in metal coordination. The kinetic characteristics and metal binding stoichiometries of the purified site-directed substitutions of each of the histidine and cysteine residues in the catalytic domain of the protein to asparagine and serine residues, respectively, were determined. These data support the hypothesis that the histidines at positions 55, 57, and 201 are coordinated to a metal ion (M1) at the active center of the enzyme and that His254 and His257 are involved in the formation of a second structural metal center (M2). These and other unidentified amino acids may participate in a co-catalytic center. Although previous solution chemical studies concluded that cysteines are not involved in metal coordination, serine substitutions for Cys59 and Cys227 do affect metal content and catalytic activity. In contrast, substitution of asparagine for His230 does not affect the metal stoichiometry, but does reduce the kcat by 10(-4), indicating that it may be directly involved in the reaction chemistry. The H201N substitution eliminates activity but maintains one molar equivalent of metal and may function as a bridging ligand.
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Department of Biochemistry and Biophysics, Texas A & M University System, College Station 77843-2128.
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Wild
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010261:Paraoxon / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D008670:Metals; D011994:Recombinant Proteins; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases; D010261:Paraoxon