Identification of functional sites in the catalytic subunit of cAMP-dependent protein kinase by differential modification and site-directed mutagenesis
In order to characterize substrate-induced conformational changes in the catalytic (C) subunit of cAMP-dependent protein kinase, the reactivity of the lysine residues towards ({sup 3}H)-acetic anhydride was determined in the absence of substrates, with MgATP bound to the enzyme, and when MgATP and an inhibitor peptide were present. A model for a portion of the ATP binding site in the C subunit was proposed based in part on the changes in lysine reactivity induced by MgATP binding to the C subunit. An intramolecular cross-link between a carboxyl group activated by dicyclohexylcarbodiimide (DCCD) and a lysine residue was circumvented by first modifying the lysine residues in the C subunit with acetic anhydride, and then labeling the modified C subunit with DCCD and ({sup 14}C)-glycine ethyl ester. Two carboxyl groups, Asp 184 and Glu 91, were labeled in the apoenzyme, but protected from modification in the presence of MgATP. The two residues accounting for the intramolecular cross-link mediated by DCCD were identified by first labeling the apoenzyme with DCCD, followed by modification of the lysine residues with ({sup 3}H)-acetic anhydride. The two residues involved in the cross-link, Asp 184 and Lys 72, are both invariant amino acids in the protein kinase family, and a potential orientation of the active site was proposed. The C subunit was modified with a water soluble carbodiimide, 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide (EDC) and ({sup 14}C)glycine ethyl ester in order to identify carboxyl groups that may interact with the basic residues of the protein substrates. Either MgATP or peptide inhibitor alone did not protect the C subunit from inhibition, but together they blocked the inactivation by EDC.
- Research Organization:
- California Univ., San Diego, CA (United States)
- OSTI ID:
- 5671579
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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STRUCTURE-ACTIVITY RELATIONSHIPS
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CROSS-LINKING
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MUTAGENESIS
RECEPTORS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
CARBON COMPOUNDS
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550201* - Biochemistry- Tracer Techniques