Cofactor and DNA interactions in EcoRI DNA methyltransferase. Fluorescence spectroscopy and phenylalanine replacement for tryptophan 183.
EcoRI DNA methyltransferase contains tryptophans at positions 183 and 225. Tryptophan 225 is adjacent to residues previously implicated in S-adenosylmethionine (AdoMet) binding and to cysteine 223, previously shown to be the site of N-ethyl maleimide-mediated inactivation of the enzyme (Reich, N. O., and Everett, E. (1990) J. Biol. Chem. 265, 8929-8934; Everett, E. A., Falick, A. M., and Reich, N. O. (1990) J. Biol. Chem. 265, 17713-17719). The fluorescence spectra of the wild-type enzyme is centered at 338 nm indicating partial tryptophan solvent accessibility. Substitution of tryptophan 183 with phenylalanine results in a 45% drop in fluorescence intensity, but no shift in lambda max. DNA binding to the wild-type methyltransferase caused an increase in the fluorescence intensity, while binding to the tryptophan 183 mutant had a quenching effect, suggesting that DNA binding induces a conformational change near both tryptophans. Binding of AdoMet and various AdoMet analogs to the wild-type methyltransferase results in no change in the fluorescence spectrum when excitation occurs at 295 nm, suggesting that no conformational change occurs, and AdoMet does not interact with either tryptophan. In contrast, quenching was observed when excitation occurred at 280 nm, suggesting that AdoMet and its analogs may be quenching tyrosine to tryptophan energy transfer. Protein-ligand complexes were titrated with acrylamide, and the data also implicate conformational changes upon DNA binding but not upon AdoMet binding, consistent with previous limited proteolysis results (Reich, N. O., Maegley, K. A., Shoemaker, D.D., and Everett, E. (1991) Biochemistry 30, 2940-2946).
Maegley
K A
KA
Department of Chemistry, University of California, Santa Barbara 93106.
Gonzalez
L
L
Smith
D W
DW
Reich
N O
NO
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D005454:Fluorescence Polarization; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics*
D000178:Acrylamides; D020106:Acrylamide; D010649:Phenylalanine; D012436:S-Adenosylmethionine; D014364:Tryptophan; D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)