Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with a water-soluble carbodiimide: identification of carboxyl groups protected by MgATP and inhibitor peptides.
The catalytic subunit of cAMP-dependent protein kinase typically phosphorylates protein substrates containing basic amino acids preceding the phosphorylation site. To identify amino acids in the catalytic subunit that might interact with these basic residues in the protein substrate, the enzyme was treated with a water-soluble carbodiimide, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), in the presence of [14C]glycine ethyl ester. Modification of the catalytic subunit in the absence of substrates led to the irreversible, first-order inhibition of activity. Neither MgATP nor a 6-residue inhibitor peptide alone was sufficient to protect the catalytic subunit against inactivation by the carbodiimide. However, the inhibitor peptide and MgATP together completely blocked the inhibitory effects of EDC. Several carboxyl groups in the free catalytic subunit were radiolabeled after the catalytic subunit was modified with EDC and [14C]glycine ethyl ester. After purification and sequencing, these carboxyl groups were identified as Glu 107, Glu 170, Asp 241, Asp 328, Asp 329, Glu 331, Glu 332, and Glu 333. Three of these amino acids, Glu 331, Glu 107, and Asp 241, were labeled regardless of the presence of substrates, while Glu 333 and Asp 329 were modified to a slight extent only in the free catalytic subunit. Glu 170, Asp 328, and Glu 332 were all very reactive in the apoenzyme but fully protected from modification by EDC in the presence of MgATP and an inhibitor peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Buechler
J A
JA
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Taylor
S S
SS
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002234:Carbodiimides / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D010455:Peptides / Q000494:pharmacology*; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D012995:Solubility; D013552:Swine
D002234:Carbodiimides; D046911:Macromolecular Substances; D010455:Peptides; D047428:Protein Kinase Inhibitors; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D005022:Ethyldimethylaminopropyl Carbodiimide