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Tyrosine 264 in the recA protein from Escherichia coli is the site of modification by the photoaffinity label 8-azidoadenosine 5'-triphosphate.

1985; Elsevier BV; Volume: 260; Issue: 18 Linguagem: Inglês

10.1016/s0021-9258(17)39230-x

1083-351X

Kendall L. Knight, Kathleen Mc Entee,

Bacterial Genetics and Biotechnology

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 ATPbinding 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 [a-32P]N3-ATP-labeled T3 1 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 [~-~~P]N~ATP-modified T31 and unmodified T3 1 using a-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.