EcoRI endonuclease. Physical and catalytic properties of the homogenous enzyme.
A procedure for large scale isolation of Escherichia coli RI endonuclease in high yield has been developed. The purified enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and analytical sedimentation. The denatured and reduced form of the enzyme has a molecular weight of 28,500 +/- 500. In solution the enzyme exists as a mixture of dimers and tetramers of molecular weights 57,000 and 114,000, respectively. We estimate the dissociation constant for tetramer to dimer transition to be less than or approximately equal to 1 x 10-7 M. Steady state kinetic analysis of the endonuclease with ColE1 DNA as substrate showed that the enzyme obeys Michaelis-Menten kinetics. At 37 degrees the turnover number is four double strand scissons per min, and the Km for ColE1 molecules is 8 x 10(-9) M. At 0 degrees the major product of endonuclease action contains only one single strand break in the RI site, and such molecules can dissociate from the enzyme. In contrast, at 30 degrees to 37 degrees, two single strand breaks are introduced into the RI sequence prior to dissociation of the enzyme. A transient enzyme-bound intermediate containing only one break in the RI site was observed in studies of a single turnover at 30 degrees. Kinetic analysis of this reaction indicates that the first break is introduced into the RI site with the first order rate constant of at least 40 min-1, while the second cleavage occurs with a rate constant of 14 min-1. Since the turnover number of the enzyme at 30 degress is only 0.72 min-1, these results indicate that the rate-limiting step is release of endonuclear from its DNA product.
D000596:Amino Acids / Q000032:analysis; D003851:Deoxyribonucleases / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight
D000596:Amino Acids; D003851:Deoxyribonucleases; D004720:Endonucleases