THE INTRINSIC ATPASE ACTIVITY OF PROTEIN-KINASE-C IS CATALYZED AT THE ACTIVE-SITE OF THE ENZYME

We recently reported that autophosphorylated protein kinase C (PKC) has an intrinsic Ca2+- and phospholipid-dependent ATPase activity and that the ATPase and histone kinase activities of PKC have similar metal-ion cofactor requirements and K(m,app)(ATP) values. We hypothesized that the intrinsic ATPase activity of PKC may represent the bond-breaking step of its protein kinase activity. The rate of the ATPase reaction is several times slower than the histone kinase reaction rate. At subsaturating concentrations, various peptide and protein substrates stimulate the ATPase reaction by as much as 1.5-fold. In contrast, non-phosphorylatable substrate analogs are not stimulatory. These observations support a mechanism of PKC catalysis in which the productive binding of phosphoacceptor substrates enhances the rate of phosphodonor substrate (ATP) hydrolysis at the active site of PKC. However, this mechanism contains an assumption that the ATPase activity of PKC is catalyzed at the active site. In fact, sequence analysis indicates that PKC contains a potential second nucleotide binding site outside of its active site. In this report, we provide a detailed analysis of the relationship between the active site of PKC and the intrinsic ATPase activity of the enzyme. We show that the regulatory and catalytic properties of the ATPase reactions of three PKC isozymes are similar, despite critical differences among the isozymes in their consensus sequences for the potential non-active-site nucleotide binding site in their catalytic domains. We also show that the ATPase and histone kinase reactions of each isozyme have similar K(m,app)(ATP) values. Furthermore, we demonstrate that an active-site-directed anti-PKC monoclonal antibody has parallel stimulatory effects on the ATPase and histone kinase activities of PKC and that monovalent salts have parallel inhibitory effects against these activities. Finally, we report that H7, which inhibits protein kinases but not other ATP-utilizing enzymes by competition with ATP, inhibits the ATPase activity of PKC with predominantly competitive kinetics. Taken together, the data presented in this report provide convincing evidence that the ATPase activity of PKC is catalyzed at the active site of the enzyme. Our results also indicate the usefulness of the ATPase reaction as a diagnostic tool in studies of the bond-breaking step of the protein kinase reaction of PKC.