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The kinetic mechanism of EcoRI endonuclease.

Publication ,  Journal Article
Wright, DJ; Jack, WE; Modrich, P
Published in: J Biol Chem
November 5, 1999

Steady-state parameters governing cleavage of pBR322 DNA by EcoRI endonuclease are highly sensitive to ionic environment, with K(m) and k(cat) increasing 1,000-fold and 15-fold, respectively, when ionic strength is increased from 0.059 to 0.23 M. By contrast, pre-steady-state analysis has shown that recognition, as well as first and second strand cleavage events that occur once the enzyme has arrived at the EcoRI site, are essentially insensitive to ionic strength, and has demonstrated that the rate-limiting step for endonuclease turnover occurs after double-strand cleavage under all conditions tested. Furthermore, processive cleavage of a pBR322 variant bearing two closely spaced EcoRI sites is governed by the same turnover number as hydrolysis of parental pBR322, which contains only a single EcoRI sequence, ruling out slow release of the enzyme from the cleaved site or a slow conformational change subsequent to double-strand cleavage. We attribute the effects of ionic strength on steady-state parameters to nonspecific endonuclease.DNA interactions, reflecting facilitated diffusion processes, that occur prior to EcoRI sequence recognition and subsequent to DNA cleavage.

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Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

November 5, 1999

Volume

274

Issue

45

Start / End Page

31896 / 31902

Location

United States

Related Subject Headings

  • Plasmids
  • Osmolar Concentration
  • Models, Chemical
  • Kinetics
  • Deoxyribonuclease EcoRI
  • DNA, Bacterial
  • Biochemistry & Molecular Biology
  • 34 Chemical sciences
  • 32 Biomedical and clinical sciences
  • 31 Biological sciences
 

Citation

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Wright, D. J., Jack, W. E., & Modrich, P. (1999). The kinetic mechanism of EcoRI endonuclease. J Biol Chem, 274(45), 31896–31902. https://doi.org/10.1074/jbc.274.45.31896
Wright, D. J., W. E. Jack, and P. Modrich. “The kinetic mechanism of EcoRI endonuclease.J Biol Chem 274, no. 45 (November 5, 1999): 31896–902. https://doi.org/10.1074/jbc.274.45.31896.
Wright DJ, Jack WE, Modrich P. The kinetic mechanism of EcoRI endonuclease. J Biol Chem. 1999 Nov 5;274(45):31896–902.
Wright, D. J., et al. “The kinetic mechanism of EcoRI endonuclease.J Biol Chem, vol. 274, no. 45, Nov. 1999, pp. 31896–902. Pubmed, doi:10.1074/jbc.274.45.31896.
Wright DJ, Jack WE, Modrich P. The kinetic mechanism of EcoRI endonuclease. J Biol Chem. 1999 Nov 5;274(45):31896–31902.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

November 5, 1999

Volume

274

Issue

45

Start / End Page

31896 / 31902

Location

United States

Related Subject Headings

  • Plasmids
  • Osmolar Concentration
  • Models, Chemical
  • Kinetics
  • Deoxyribonuclease EcoRI
  • DNA, Bacterial
  • Biochemistry & Molecular Biology
  • 34 Chemical sciences
  • 32 Biomedical and clinical sciences
  • 31 Biological sciences