Reaction kinetics of some important site-specific endonucleases.
AUTOR(ES)
Hinsch, B
RESUMO
Reaction kinetics of the site-specific endonucleases BamHI, BgIII, C1aI, EcoRI, HpaII, PstI, SaII, SmaI, and XorII were investigated employing some frequently used substrates. Six of these enzymes could be analyzed under steady-state conditions. Kinetic data were obtained from progress curves applying an integrated Michaelis-Menten equation. KM ranged from 4 x 10(-9) M to 4 x 10(-11) M. Activities also spanned two orders of magnitude. In the case of C1aI the analysis of the pre-steady-state kinetics ("burst reaction") allowed the assessment of several rate constants. The rate-limiting step is the very slow dissociation of the enzyme-product complex (0.22 min(-1)). This complex is formed from the enzyme-bound nicked intermediate at a rate of 1.7 min(-1). The introduction of the first cut is again faster by a factor of about 6. SmaI and XorII resembled C1aI in their kinetics. The burst reaction can be used for the easy and unambiguous determination of molar concentrations of site-specific endonucleases in any preparation, which is free of non-specific DNases.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=327339Documentos Relacionados
- Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases.
- Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.
- Site-specific methylation: effect on DNA modification methyltransferases and restriction endonucleases
- Biochemical and genetic properties of site-specific restriction endonucleases in Bacillus globigii.
- Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.
