Modification of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity labels related to peptide substrates.
The modification and concomitant inactivation of the catalytic subunit of bovine heart cAMP-dependent protein kinase with affinity analogs of peptide substrates potentially capable of undergoing disulfide interchange with enzyme-bound sulfhydryl groups have been used to probe the active site associated with peptide binding. The regeneration of catalytic activity on treatment of the modified enzymes with dithiothreitol and the observation that prior reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) blocks the modification of the kinase by these reagents are consistent with the proposal that only thiol residues are reacting. The affinity analog Leu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly, 1, and the closely related peptide AcLeu-Arg-Arg-Ala-Cys(3-nitro-2-pyridinesulfenyl)-Leu-Gly-OEt, 3, react with a single sulfhydryl as shown by the stoichiometry of the release of the 3-nitro-2-pyridinesulfenyl group and the amount of label incorporated in the enzyme when the radioactively labeled peptide analog of 3 (peptide 4) is employed as the modifying agent. The kinetics of the reaction of 1 with 4.3 microM catalytic subunit was monophasic (employing substrate in excess conditions), yielding an apparent value of KI of approximately 40 microM and a k2 value of approximately 0.25 s-1. The low value of the observed KI, together with the observation that protein kinase substrates inhibit the modification reactions, suggest strongly that the cysteine residue undergoing reaction is in the vicinity of the active site. By trypsin-catalyzed degradation and identification of the peptide segment modified by covalent attachment of the peptide portion of the radioactive analog 4, the single cysteine modified was identified as cysteine-198.
D000345:Affinity Labels / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology*; D009842:Oligopeptides / Q000494:pharmacology*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D046911:Macromolecular Substances; D009842:Oligopeptides; D000242:Cyclic AMP; D011494:Protein Kinases