Covalent modification with concomitant inactivation of the cAMP-dependent protein kinase by affinity labels containing only L-amino acids.

Affinity labels for proteins that process other proteins (e.g. proteinases and protein kinases) are an amalgam of two components, an active site-directed peptide carrier and a non-peptidic electrophilic appendage. We have synthesized several affinity labels for the cAMP-dependent protein kinase that are composed solely of L-amino acids and therefore contain only functionality present in naturally occurring proteins. We have found that 2 adjacent cysteine residues, when covalently linked via a peptide bond and an intramolecular disulfide loop (abbreviated as Cys<-->Cys), serves as a potent electrophile. The peptides Leu-Arg-Arg-Cys<-->Cys-Leu-Gly (1), Leu-Arg-Arg-Ala-Cys<-->Cys-Gly (2), and Leu-Arg-Arg-Ala-Ala-Cys<-->Cys-Gly (3) inactivate the cAMP-dependent protein kinase in a time-dependent fashion. Since dialysis does not restore activity, but dithiothreitol does, this strongly suggests that covalent modification of the target enzyme has occurred at a cysteine residue. Although there are 2 cysteine moieties contained within the protein kinase, the 14C-acetylated affinity labels modify the enzyme only once. In addition, since ATP blocks inactivation of the protein kinase, this implies that it is the active site cysteine residue (Cys-199) that has undergone covalent modification. Based on the KI(inact) values obtained from inactivation kinetics, we conclude that the optimal site on the affinity label for the electrophilic Cys<-->Cys is 1 amino acid removed from the 2 arginine residues (i.e. 2). In addition, the efficacy of these inhibitors is also dependent upon the size of the disulfide ring. The eight-membered disulfide ring-containing peptides 1-3 are relatively poor affinity labels compared to the 12-membered ring-containing inhibitor, [formula: see text]