Localization of the High‐Affinity ATP Site in Adenosine‐3′:5′‐monophosphate‐Dependent Protein Kinase Type I

Abstract

8-Azido-adenosine 5'-triphosphate (n8(3)ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3':5'-monophosphate (cAMP). It competes with ATP for the high-affinity ATP site in the undissociated form of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction. n8(3)ATP incorporated into the holoenzyme is covalently bound irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high-affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n8(3)ATP is bound to the catalytic subunit. After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [gamma-32P]n8(3)ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low-affinity catalytically active ATP site into a high-affinity inactive site.