Restriction enzyme HhaII catalytic rate (Kcat)

Value 0.2 min^-1
Organism Unspecified
Reference Kaddurah-Daouk R, Cho P, Smith HO. Catalytic properties of the HhaII restriction endonuclease. J Biol Chem. 1985 Dec 5260(28):15345-51. p.15349 right column, second paragraphPubMed ID2999112
Method Researchers used two different assays in this work. One assay employs agarose gel electrophoresis to separate DNA product bands produced by cleavage of plasmid pSKll which contains a single HhaII site, and the amount of DNA in each band is determined by ethidium bromide staining and densitometry. The second assay employs pSK11 DNA specifically labeled with 32P-atoms within the site. HhaII cleavage exposes the labeled phosphoryl groups which are then released as inorganic phosphate by phosphatase action. Released radioactive phosphate is then quantitated by thin layer chromatography.
Comments The rate constants for the cleavage of the first and second phosphodiester bonds in a site are a function of enzyme concentration up to the point where all sites are saturated with enzyme. At near-saturating conditions and irrespective of the mode of mixing, the cleavage of each strand follows a first order reaction with k1=~0.8 (0.76) and k2=~0.2 single strand scissions/min.
Entered by Ben Marks
ID 101632