Stringent and relaxed specificities of TaqI endonuclease: interactions with metal cofactors and DNA sequences.
We have studied the roles of metal cofactors Mg2+ and Mn2+ in modulating substrate specificities during the enzymatic cycle of TaqI endonuclease using steady state and single-turnover kinetics. In the presence of Mg2+, stringent discrimination of TaqI against single base-pair changes (star sites) is manifested by the loss of tight, specific binding in the early stage of the enzymatic cycle. In the presence of Mn2+, relaxed specificity for a star site sequence is attributed to formation of three distinct classes of the ternary complexes: the highly activated TaqI-cognate-Mn2+ complex; the partially activated TaqI-star-Mn2+ complex; and the ground state, inactive TaqI-nonspecific-Mn2+ complex. In addition to a high affinity for a TaqI-DNA complex, Mn2+ also binds to TaqI in a DNA-independent fashion. This may facilitate enzyme activation, which could account for the observed relaxation in substrate specificity. Thus, the TaqI-DNA-Mn2+ complex could be formed by either of two pathways: TaqI binding to DNA followed by the binding of Mn2+ or TaqI first binding to Mn2+ followed by the addition of DNA. The inactive, nonspecific TaqI-star-Mg2+ complex virtually prohibits transition state interactions, but a TaqI-star-Mn2+ complex attains a measurable single-turnover rate. In the late stages of the enzymatic cycle, high affinity of Mn2+ to a TaqI-DNA complex and to the TaqI enzyme may also account for a slower rate of product release.
Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College, New York, New York 10021.
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D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D008345:Manganese; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases; D008274:Magnesium