Stringent and relaxed specificities of TaqI endonuclease: interactions with metal cofactors and DNA sequences. Academic Article uri icon

Overview

abstract

  • 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.

publication date

  • February 21, 1995

Research

keywords

  • Deoxyribonucleases, Type II Site-Specific
  • Magnesium
  • Manganese

Identity

Scopus Document Identifier

  • 0028985732

Digital Object Identifier (DOI)

  • 10.1021/bi00007a023

PubMed ID

  • 7857938

Additional Document Info

volume

  • 34

issue

  • 7