Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase.
The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation [Toner-Webb, J., & Taylor, S. S. (1987) Biochemistry 26, 7371]. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [14C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.(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 / Q000494:pharmacology; D000818:Animals; D001224:Aspartic Acid; D002250:Carbon Radioisotopes; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D000242:Cyclic AMP / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005971:Glutamates; D018698:Glutamic Acid; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine; D014357:Trypsin
D002250:Carbon Radioisotopes; D005971:Glutamates; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D001224:Aspartic Acid; D018698:Glutamic Acid; C022010:glycine ethyl ester; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D002918:Chymotrypsin; D014357:Trypsin; D005998:Glycine