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
- Univ. of California, San Diego, La Jolla (USA)
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 ({sup 14}C)glycine ethyl ester. Several carboxyl groups in the free catalytic subunit were radiolabeled after the catalytic subunit was modified with EDC and ({sup 14}C)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 Glue 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. In any case, the results indicate a dramatic change in the accessibility of this region of the catalytic subunit in the presence and absence of substrates.
- OSTI ID:
- 6897357
- Journal Information:
- Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:7; ISSN 0006-2960; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
AMINO ACID SEQUENCE
AMINO ACIDS
AMP
ASPARTIC ACID
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CATALYSIS
CHEMICAL REACTIONS
ENZYMES
ESTERS
GLUTAMIC ACID
GLYCINE
IMIDES
LABELLED COMPOUNDS
LABELLING
MAGNESIUM COMPOUNDS
MOLECULAR STRUCTURE
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
PURIFICATION
TRANSFERASES