Affinity labeling of the ATP-binding site of type II calmodulin-dependent protein kinase by 5'-p-fluorosulfonylbenzoyl adenosine.
Modification of the type II calmodulin-dependent protein kinase by 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) resulted in a time-dependent inactivation of the enzyme. The reaction followed pseudo-first-order kinetics and showed a nonlinear dependence on reagent concentration. The rate of inactivation was sensitive to Mg2+- and calmodulin-induced conformational changes on the enzyme. However, the enhancing effects of these ligands were not additive; indeed, the kinetic parameters of the Mg2+-stimulated inactivation reaction with FSBA (Kinact = 2.4 mM; kappa max = 0.12 min-1) were almost unaffected by the simultaneous addition of calmodulin (Kinact = 1.5 mM; kappa max = 0.086 min-1). Protection from inactivation by FSBA was provided by Mg2+-ADP which is consistent with modification of the catalytic site. An analysis of the protective effect of Mg2+-ADP in the absence (Kd = 590 microM) and presence (Kd = 68 microM) of calmodulin demonstrated that binding of the modulator protein to the enzyme increases the affinity of the protein kinase for nucleotides. Modification by FSBA resulted in labeling of both Tyr and Lys residues but only labeling of Lys was decreased by Mg2+-ADP which is consistent with the hypothesis that a conserved Lys residue is important in nucleotide binding to the protein kinase. However, the kinetic results of the inactivation reaction suggest that this Lys is not involved in mediating the calmodulin-promoted increase in the affinity of the enzyme for Mg2+-nucleotide complexes.
King
M M
MM
Department of Chemistry, Ohio State University, Columbus 43210.
Shell
D J
DJ
Kwiatkowski
A P
AP
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels / Q000494:pharmacology*; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D007700:Kinetics; D008024:Ligands / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*
D000345:Affinity Labels; D008024:Ligands; D047428:Protein Kinase Inhibitors; D000244:Adenosine Diphosphate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D008274:Magnesium; D000241:Adenosine