Affinity labeling of the ATP binding site of bovine lung cyclic GMP-dependent protein kinase with 5'-p-fluorosulfonylbenzoyladenosine.
Bovine lung cyclic GMP-dependent protein kinase was covalently labeled with the ATP analog, 5-p-fluorosulfonylbenzoyladenosine. The inactivation reaction was pseudo-first order. The rate of kinase sulfonation exhibited saturation kinetics indicative of a rapid reversible binding of the reagent prior to enzyme modification. The enzyme could be protected by MgATP, MgADP, and Mg-adenylylimidodiphosphate but not by a synthetic peptide substrate. Cyclic GMP when bound to the kinase did not influence the rate of labeling. The reagent demonstrated competitive inhibition with respect to MgATP; the Ki was found to be 0.82 mM. Magnesium and cobalt ions when included in the reaction mixture accelerated the inactivation rate up to severalfold. Addition of basic polypeptides such as mixed histone, protamine sulfate, or poly-L-lysine HBr also markedly accelerated the sulfonation rate. Inactivation of the kinase with 5-'fluorosulfonyl[3H]benzoyladenosine resulted in a linear relationship between the residual phosphotransferase activity and the incorporation of up to 0.9 mol of reagent/mol of monomer.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology*; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D002614:Chelating Agents; D003035:Cobalt; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D006152:Cyclic GMP; D008274:Magnesium; D000241:Adenosine