Asp537 and Asp812 in bacteriophage T7 RNA polymerase as metal ion-binding sites studied by EPR, flow-dialysis, and transcription.
Asp537 and Asp812 are essential in the catalytic mechanism of T7 RNA polymerase. The mutants D537N and D812N have no detectable activity whereas the mutants D537E and D812E have significantly reduced activity relative to the wild-type. The hypothesis that these two amino acids act as metal-binding ligands has been tested using EPR with Mn2+ as the metal ion. Mn2+ is able to substitute for Mg2+ in transcription by T7 RNAP on templates containing the T7 promoter. Mg2+ and Mn2+ compete for binding sites, with the former having lower affinity. Mn2+ binding to the wild-type enzyme and the mutants D537N, D812N, D537E, D812E, and Y649F was measured over the concentration range of 25 microM to 1.5 mM. The data were analyzed by nonlinear least-squares fits to the binding isotherms, and the analysis gave approximately two Mn(2+)-binding sites in all cases and a Kd for the wild-type of approximately 340 microM. The Kd value for the mutant Y639F, in which Asp537 and Asp 812 are not mutated, is comparable to the value for the wild-type. Mn2+ binding to the double mutants, D537N/D812N and D537E/D812E, appears to be nonspecific. The Kd values of the Asp-->Asn mutants are only 2-5 times larger than the value for the wild-type, in contrast to the drastic diminution of enzymatic activity in the mutants. The geometry of metal binding to these Asp residues may be crucial in determining the catalytic competence. Mn2+ binding to the wild-type enzyme in the presence of nucleotides, measured by flow dialysis, is characterized by two Mn(2+)-binding sites with a Kd value of ca. 150 microM. The similarity in values of Kd with and without nucleotide suggests that nucleotides do not have a drastic effect on Mn2+ binding. Our results indicate that monodentate carboxylate oxygens of both conserved Asp residues bridge the two metal ions.
Woody
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Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins 80523, USA.
Eaton
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Osumi-Davis
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Woody
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D000595:Amino Acid Sequence; D001224:Aspartic Acid; D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D003956:Dialysis; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; D001224:Aspartic Acid; D008345:Manganese; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium