Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with acetic anhydride: substrate-induced conformational changes.
In order to identify regions that are sensitive to substrate-induced perturbations, the catalytic subunit of cAMP-dependent protein kinase was differentially labeled with [3H]acetic anhydride. Treatment of the catalytic subunit with acetic anhydride in the absence of substrates led to the irreversible inhibition of activity, and MgATP protected against inactivation. After development of a purification protocol for the lysine-containing peptides, the reactivity of each lysine in the native enzyme was calculated. The reactivity profile of lysines in the apoenzyme revealed three distinct regions. In general, the lysines within the amino-terminal segment (residues 1-83) and the carboxy-terminal segment (192-345) were relatively reactive. In contrast, the five lysines in the middle of the protein (Lys-92, -105, -111, -168, and -189) were very unreactive, indicating that these groups are sequestered from the aqueous solvent. The reactivity of each lysine was then determined in the presence of MgATP and in the presence of MgATP and a 20-residue inhibitor peptide. Most of the substrate-induced changes in lysine reactivity were localized in the amino-terminal segment, while the reactivities of lysines in the carboxy-terminal region were not altered significantly by MgATP or inhibitor peptide. MgATP affords substantial protection to three residues in particular. Lys-72, predicted previously to be essential for nucleotide binding was relatively reactive in the apoenzyme, whereas labeling was nearly abolished in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Buechler
J A
JA
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Vedvick
T A
TA
Taylor
S S
SS
D000085:Acetates / Q000378:metabolism*; D000089:Acetic Anhydrides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D008239:Lysine; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine; D014316:Tritium
D000085:Acetates; D000089:Acetic Anhydrides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014316:Tritium; C031800:acetic anhydride; D011494:Protein Kinases; D008239:Lysine