Affinity labeling of a tyrosine residue in the ATP binding site of the recA protein from Escherichia coli with 5'-p-fluorosulfonylbenzoyladenosine.
We have covalently modified the recA protein from Escherichia coli with the adenine nucleotide analog 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA). The rate at which the protein is modified shows a sigmoidal dependence on the concentration of 5'-FSBA suggesting that binding of the analog is characterized by positive cooperativity. Covalent modification of the protein results in irreversible inactivation of its single-stranded DNA-dependent ATPase activity such that 100% inactivation is achieved when 25% of the enzyme monomers have been modified. Attachment of 5'-FSBA is specific for the ATP-binding site of recA protein as judged by the following criteria: (i) attachment of the affinity label to the protein appears to saturate at 1 mol of 5'-FSBA/mol of protein; (ii) binding of 5'-FSBA to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine-5'-O-(thiotriphosphate), ADP, UTP, and GTP, but not by adenosine; (iii) attachment of 5'-FSBA to the protein occurs at a single site as determined by high pressure liquid chromatography peptide separation. Following trypsin digestion of recA protein that had been covalently modified with [3H]5'-FSBA we isolated a single labeled peptide (T31) containing the exclusive site of 5'-FSBA attachment. A secondary proteolytic digestion was performed on both 5'-FSBA modified T31 and unmodified T31 using Staphylococcus aureus V8 protease, and by comparison of the amino acid compositions of the resulting peptides we identified Tyr-264 as the exclusive site of 5'-FSBA attachment in recA protein.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*; D014357:Trypsin; D014443:Tyrosine
D000345:Affinity Labels; D000596:Amino Acids; D010446:Peptide Fragments; D014443:Tyrosine; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D014357:Trypsin; D000241:Adenosine