Identification of peptides involved in S-adenosylmethionine binding in the EcoRI DNA methylase. Photoaffinity laveling with 8-azido-S-adenosylmethionine.
The Mr 38,050 monomeric EcoRI DNA methylase is part of a bacterial restriction-modification system. The methylase transfers the methyl group from S-adenosylmethionine (AdoMet) to the second adenine in the double-stranded DNA sequence 5'-GAATTC-3'. We have used the radiolabeled photoaffinity analog 8-azido-S-adenosylmethionine (8-N3-AdoMet) to identify peptides at the AdoMet binding site in the binary methylase-cofactor analog complex. The dissociation constants in the absence of DNA for the analog and AdoMet are 12.9 and 4.8 microM, respectively. The apparent kcat and Km values, obtained with the double-stranded DNA substrate 5'-CGCGAATTCGCG-3', are 5.0 s-1 and 0.710 microM (8-N3-AdoMet) and 4.3 s-1 and 0.335 microM (AdoMet). Photolabeling by 8-N3-AdoMet occurs upon irradiation with ultraviolet light and is inhibited by AdoMet. Digestion of the adducted methylase with subtilisin generated several radiolabeled peptides. Peptide sequencing from independent photolabeling experiments revealed two radiolabeled peptides containing amino acids 206-212 and 213-221. Instability of the adducted peptides precluded assignment of modified amino acids.
Reich
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Chemistry Department, University of California, Santa Barbara 93106.
Everett
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D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010782:Photolysis; D011485:Protein Binding; D012436:S-Adenosylmethionine / Q000378:metabolism*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000138:chemical synthesis / Q000378:metabolism*; D013379:Substrate Specificity
D000345:Affinity Labels; D012436:S-Adenosylmethionine; D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)