Tyrosine 264 in the recA protein from Escherichia coli is the site of modification by the photoaffinity label 8-azidoadenosine 5'-triphosphate.
The photoaffinity label 8-azidoadenosine 5'-triphosphate (N3-ATP) was used to covalently modify the recA protein from Escherichia coli within its ATP-binding site. We have previously demonstrated that N3-ATP modification of recA protein is specific for the ATP-binding site and have isolated a unique tryptic peptide (T31), spanning residues 257-280, that contains the exclusive site of attachment of this ATP analog (Knight, K. L., and McEntee, K. (1985) J. Biol. Chem. 260, 867-872). We performed a secondary proteolytic digestion of the [alpha-32P]N3-ATP-labeled T31 peptide using Staphylococcus aureus V8 protease and purified the resulting peptide fragments by high-pressure liquid chromatography (HPLC). Based on a comparison of the amino acid compositions of all purified fragments and sequence analysis of one labeled fragment we determined that Tyr-264 is the exclusive site of N3-ATP attachment in recA protein. Photoaffinity labeling of recA protein was also performed in the presence of single-stranded DNA. Following trypsin treatment and separation of peptides by HPLC we showed that tryptic peptide T31 contained the exclusive site of N3-ATP attachment. A secondary proteolytic digestion was performed on both [alpha-32P]N3ATP-modified T31 and unmodified T31 using alpha-chymotrypsin. Comparison of the HPLC profiles and amino acid compositions of the resulting fragments was consistent with Tyr-264 as the exclusive site of N3-ATP attachment to recA protein.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D001386:Azides / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000378:metabolism*; D014357:Trypsin; D014443:Tyrosine
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D014443:Tyrosine; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D014357:Trypsin