Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide
The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent K/sub i/ of 60 ..mu..M. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD were made. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys. Two of these carboxyl groups, Asp-166 and Asp-184, are conserved in all protein kinases, including oncogene transforming proteins and growth factor receptors, and thus are likely to play an essential role.
- Research Organization:
- Univ. of California, San Diego, La Jolla
- OSTI ID:
- 5437744
- Journal Information:
- Biochemistry; (United States), Vol. 26:23
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
IMIDES
BIOLOGICAL EFFECTS
PHOSPHOTRANSFERASES
BIOCHEMICAL REACTION KINETICS
INHIBITION
AMINO ACID SEQUENCE
AMP
ATP
CARBON 14 COMPOUNDS
CATIONS
ELECTROPHORESIS
LIQUID COLUMN CHROMATOGRAPHY
MAGNESIUM COMPOUNDS
PH VALUE
PHOSPHORUS 32
TRITIUM COMPOUNDS
ALKALINE EARTH METAL COMPOUNDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHARGED PARTICLES
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ENZYMES
IONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MOLECULAR STRUCTURE
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
RADIOISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
TRANSFERASES
550201* - Biochemistry- Tracer Techniques