Open AccessCation Dependence of Restriction Endonuclease Eco RI Activity
Author(s) -
WOODHEAD J. Lesley,
BHAVE Neetin,
MALCOLM Alan D. B.
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb05237.x
Subject(s) - ecori , divalent , chemistry , restriction enzyme , ionic radius , ionic strength , enzyme , dna , crystallography , stereochemistry , biochemistry , ion , aqueous solution , organic chemistry
Restriction endonuclease Eco RI cleaves the DNA sequence under optimum digestion conditions. A variation in pH and ionic strength can result in Eco RI * activity when is cut. A divalent cation, usually Mg 2+ , is required for enzyme activity, though Mn 2+ can also be used. Eight different cations with ionic radius/charge ratios similar to Mg 2+ were tested and CO 2+ and Zn 2+ were also found to act as cofactors for Eco RI. A comprehensive study has been made of the effect of NaCl and pH on the Eco RI/ Eco RI * transition in the presence of the above four cations. Generally, a decrease in NaCl and/or an increase in pH caused a decrease in enzyme specificity. The changeover depended on the cation. They may be placed in order of their ability to increase Eco RI specificity thus: Co 2+ > Zn 2− > Mg 2+ > Mn 2+ . The K m of Eco RI for ColE1 DNA, in the presence of CO 2+ , was found to be 0.4 nM, compared to 3 nM with Mg 2+ , whereas the turnover was only one double‐stranded scission/min with Co 2+ compared to eight/min with Mg 2+ . The implications of all these findings on the enzyme's mechanism are discussed.