ATPASE-PROMOTING DEAD-END INHIBITORS OF THE CAMP-DEPENDENT PROTEIN-KINASE

The cAMP-dependent protein kinase is a bifunctional enzyme, catalyzing the phosphorylation of the serine and threonine residues in peptides and proteins (kinase activity) as well as the phosphorylation of water (ATPase activity). We have found that several peptides, which serve a s inhibitors of the kinase reaction, will either maintain or enhance t he ATPase reaction catalyzed by the enzyme. Positively charged dipepti des (e.g. Arg-Arg), as well as small guanidino-containing compounds (e .g. guanethidine) block protein kinase activity yet enhance ATPase act ivity up to 3.5-fold over that exhibited by the enzyme in the absence of these compounds. In contrast, several non-phosphorylatable peptides , whose primary sequences are based on that of a known substrate (i.e. Leu-Arg-Arg-Ala-Ser-Leu-Gly), such as Leu-Arg-Arg-Ala-Ala-Leu-Gly, Le u-Arg-Arg-Ala-Phe-Leu-Gly, and Leu-Arg-Arg-Ala-Tyr-Leu-Gly, have littl e or no effect on the rate of the kinase-catalyzed hydrolysis of ATP. An exception to the latter observation is Leu-Arg-Arg-Ala-Cys-Leu-Gly, a cysteine-containing peptide that promotes the protein kinase-cataly zed ATPase reaction by 2.2-fold. We have also found that peptides that possess relatively large amino acid side chain moieties immediately f ollowing the arginine dyad (i.e. such as Phe, Tyr, Cys, or Asn at Xaa in Leu-Arg-Arg-Xaa-Ala-Leu-Gly) sharply reduce the rate of enzyme-cata lyzed ATP hydrolysis. This suggests that in the presence of peptides c ontaining an -Arg-Arg-Ala- sequence, the enzyme-bound gamma-phosphate of ATP is relatively accessible to water. In contrast, when the the la tter alanine moiety is replaced by a larger residue, access by water t o ATP appears to be hindered. These results indicate that certain stru ctural features associated with the substrate or substrate analog have a profound influence on the manner by which these species interact wi th the protein kinase. Furthermore, the work described herein demonstr ates that it is possible to block the physiologically important kinase reaction and simultaneously promote the energetically wasteful ATPase reaction.