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Nucleic Acids Research, 1981, Vol. 9, No. 13 3159-3174
© 1981

ENZYMOLOGY

Reaction kinetics of some important site-specific endonucleases

Bernhard Hinsch and Maria-Regina Kula

Gesellschaft für Biotechnologische Forschung mbH. Mascheroder Weg 1, D-3300 Braunschweig-Stöckheim, GFR

Received April 13, 1981. Reaction kinetics of the site-specific endonucleases BamHI, BgIII, ClaI, EcoRI, HpaII, PstI, SalI, 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 · 10–9 M to 4 · 10–11 M. Activities also spanned two orders of magnitude. In the case of ClaI 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 ClaI 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.


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