Affinity labeling of catalytic subunit of bovine heart muscle cyclic AMP-dependent protein kinase by 5'-p-fluorosulfonylbenzoyladenosine.
Incubation of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase led to the formation of an inactive enzyme irreversibly modified with approximately one mol of reagent per mol of subunit. The inactivation reaction followed pseudofirst order kinetics. The rate of inactivation at various reagent concentrations exhibited saturation kinetics implying that the reagent reversibly binds to the enzyme prior to inactivation. The addition of MgATP, MgADP, or MgAMP-PNP to the reaction mixture fully protected the enzyme from inactivation by 5'-p-fluorosulfonylbenzoyladenosine. The reagent was demonstrated to be a competitive inhibitor of MgATP with a Ki of 0.235 mM. Metal-free nucleotides were without effect upon the reaction rate while metal ions alone accelerated the inactivation rate up to 7-fold. The inclusion of casein or synthetic peptide substrate in the incubation mixture did not affect the reaction kinetics. Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the kinase subunit exhibits all of the characteristics of affinity labeling of the MgATP-binding site.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D047428:Protein Kinase Inhibitors; D013379:Substrate Specificity
D000345:Affinity Labels; D046911:Macromolecular Substances; D047428:Protein Kinase Inhibitors; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000242:Cyclic AMP; D008274:Magnesium; D000241:Adenosine