Exploring the catalytic center of TaqI endonuclease: rescuing catalytic activity by double mutations and Mn2+.
TaqI is a metal-dependent endonuclease that recognizes T(downward arrow)CGA, with the arrow indicating the cleavage site. Mutations at K158 render the enzyme inactive and mutations at K157 significantly reduce DNA cleavage activity (W. Cao and F. Barany (1998) J. Biol. Chem. 273, 33002-33010). Aspartate, glutamate, and histidine substitutions were made at K158 in the wild-type and K157S mutant TaqI endonuclease to understand the functional organization of the active site. None of the mutants was active with Mg(2+), but the DNA cleavage activities were partly rescued by Mn2+ for K157S-K158E and K157S-K158H mutants. The rescuing effects were observed with Mn2+ but not with other divalent cations. K157S-K158E required higher Mn2+ concentrations than the wild-type enzyme for DNA cleavage activity, suggesting that a Mn2+ ion is weakly bound at the 158 position. The need to neutralize K157 to recover the catalytic activity of K158E and K158H indicates that K158 and K157 may interact functionally. In analogy with EcoRV, Ca2+ stimulated Mn2+-mediated cleavage for the wild-type TaqI, suggesting the existence of at least two metal ions at the catalytic center. A catalytic mechanism involving two metal ions and the K157-K158 pair is proposed for TaqI endonuclease.
Department of Microbiology and Immunology, Hearst Microbiology Research Center and Strang Cancer Prevention Center, The Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, P.O. Box 62, New York, NY 10021, USA. wgc@clemson.edu
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D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D002413:Cations, Divalent; D004249:DNA Damage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008345:Manganese / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids
D002413:Cations, Divalent; D008345:Manganese; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases; D002118:Calcium