Conformation-sensitive modification of the type II calmodulin-dependent protein kinase by phenylglyoxal.
Chemical modification by phenylglyoxal was used to investigate relationships between the structure, function, and regulation of the type II calmodulin-dependent protein kinase. Modification of the protein kinase by phenylglyoxal resulted in specific labeling of one distinct site, most likely an important arginine residue, with concomitant inactivation of the enzyme. Labeling and inactivation of the protein kinase was prevented by Mg2+-ADP which suggests that modification occurred at, or in close proximity to, its nucleotide-binding pocket. Half-maximal protection by Mg2+-ADP was enhanced by calmodulin which decreased the K0.5 for ADP from 540 to 61 microM. This response of the enzyme to calmodulin indicates that the modulator protein increases the affinity of the protein kinase for nucleotides. Inactivation of the enzyme by phenylglyoxal was dependent on the presence of Mg2+ or Ca2+/calmodulin, and further enhanced by the simultaneous addition of these effectors to the reaction. The Mg2+ effect is indicative of binding of this divalent metal ion to the protein kinase even in the absence of calmodulin and nucleotides. The stimulation of the modification reaction by calmodulin indicates an increase in the reactivity or accessibility of the modified residue in response to calmodulin-regulated conformational changes on the enzyme. The calmodulin-induced changes observed in this study may play important roles in the molecular mechanisms of activation of the type II calmodulin-dependent protein kinase.
King
M M
MM
Department of Chemistry, Ohio State University, Columbus 43210.
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010658:Phenylglyoxal / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects
D000447:Aldehydes; D002147:Calmodulin; D008274:Magnesium; D010658:Phenylglyoxal; D002118:Calcium