The kinetic mechanism of EcoRI endonuclease.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Wright, DJ; Jack, WE; Modrich, P

Published Date

  • November 5, 1999

Published In

Volume / Issue

  • 274 / 45

Start / End Page

  • 31896 - 31902

PubMed ID

  • 10542216

Pubmed Central ID

  • 10542216

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.274.45.31896

Language

  • eng

Conference Location

  • United States