Changing the hydrogen-bonding potential in the DNA binding site of EcoRI by site-directed mutagenesis drastically reduces the enzymatic activity, not, however, the preference of this restriction endonuclease for cleavage within the site-GAATTC-.
According to the X-ray structure analysis of an EcoRI-oligodeoxynucleotide complex [McClarin et al. (1986) Science 234, 1526], sequence specificity is mediated by 12 hydrogen bonds, 6 from each of the two identical subunits of the dimeric enzyme to the recognition site -GAATTC-: Arg200 forms two hydrogen bonds with guanine, while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. Changing the hydrogen-bonding potential at the recognition site without perturbing the rest of the interface should lead to the recognition of degenerate sequences [Rosenberg et al. (1987) in Protein Engineering (Oxender, D. L., & Fox, C. F., Eds.) pp 237-250, Liss, New York]. We have shown previously that replacing Glu144 by Gln and Arg145 by Lys affects the activity of the enzyme, not, however, its specificity [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. We show now that also the mutation of Arg200 to Lys, the double mutation Glu144Arg145 to GlnLys, and the triple mutation Glu144Arg145Arg200 to GlnLysLys do not lead to a detectable degeneracy of the specificity of cleavage by EcoRI but significantly impair the catalytic activity of this enzyme. A detailed analysis of the steady-state kinetics of cleavage of pUC8 DNA and a tridecadeoxynucleotide substrate demonstrates that the reduction in activity for all DNA binding site mutants investigated so far is mainly due to a decrease in kcat, with the exception of the Arg200 to Lys mutant, which is only impaired in its KM.(ABSTRACT TRUNCATED AT 250 WORDS)
Alves
J
J
Zentrum Biochemie, Medizinische Hochschule Hannover, West Germany.
Rüter
T
T
Geiger
R
R
Fliess
A
A
Maass
G
G
Pingoud
A
A
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; D015246:Deoxyribonuclease EcoRI