Peptide-based photoaffinity label of the catalytic subunit of the cyclic AMP-dependent protein kinase

A photoaffinity label of the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid L-p-benzoyl-Phe. The amino acids L- and D-p-benzoyl-Phe were synthesized from p-aminobenzophenone. Using solid-phase peptide synthesis, these were incorporated into the cAMP-dependent kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, with the amino acid enantiomers replacing the phosphorylatable Ser. The diastereomeric peptides were separated by reverse phase HPLC. The peptide substrate analogs were tested as competitive inhibitors of the catalytic subunit of the cAMP-dependent protein kinase from bovine heart. They performed equally well, with K/sub i/'s on the order of 100μM. However when photolyzed in the presence of the enzyme, it was found that only the peptide containing L-benzoyl-Phe caused photoinactivation. The photoinactivation appeared to be time- and concentration-dependent. A solution of 2μM enzyme retained only 10% activity after 2 minutes of photolysis with 100μM L-benzoyl-Phe peptide; in contrast, the enzyme was 90% active after photolysis with the D-benzoyl-Phe peptide under identical conditions. Radiolabelled L-benzoyl-Phe peptide was used to establish the binding stoichiometry of peptide to enzyme; these results showed that the photoaffinity labelling was specific (approximately 1:1). Experiments are currently being performed to determine the site of labelling on the kinase