Dicyclohexylcarbodiimide cross-links two conserved residues, Asp-184 and Lys-72, at the active site of the catalytic subunit of cAMP-dependent protein kinase.
In the absence of MgATP, the catalytic subunit of cAMP-dependent protein kinase is irreversibly inhibited by the hydrophobic carbodiimide dicyclohexylcarbodiimide, and this inhibition is most likely due to the formation of a cross-link between a carboxyl group and a lysine residue in the active site (Toner-Webb & Taylor, 1987). In order to identify these cross-linked residues, the catalytic subunit was modified by dicyclohexylcarbodiimide and then treated with acetic anhydride and digested with trypsin. The resulting peptides were resolved by high-performance liquid chromatography. One major absorbing tryptic peptide and one smaller peptide consistently and reproducibly showed a decrease in absorbance after the catalytic subunit had been treated with DCCD. These peptides correspond to residues 166-190 and 57-93, respectively. A unique peptide was isolated from the modified catalytic subunit, and the sequence of this peptide established that the cross-linking occurred between Asp-184 and Lys-72. The cross-linking of these two residues, which were both identified previously as essential residues, confirms the likelihood that each plays a role in the functioning of this enzyme. The fact that Asp-184 and Lys-72 appear to be invariant in all protein kinases further supports the hypothesis that these two residues, located close to one another at the active site of the enzyme, play essential roles in catalysis.
Buechler
J A
JA
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Taylor
S S
SS
D000255:Adenosine Triphosphate; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D001665:Binding Sites; D002234:Carbodiimides / Q000494:pharmacology*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D004024:Dicyclohexylcarbodiimide; D001216:Asparagine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D008239:Lysine