Identification of amino acid residues involved in substrate recognition by the catalytic subunit of bovine cyclic AMP dependent protein kinase: peptide-based affinity labels.
Two peptide-based affinity inactivators Ac-Leu-(BrAc)Orn-Arg-Ala-Ser-Leu-Gly (4) and Ac-Leu-Arg-(BrAc)Orn-Ala-Ser-Leu-Gly (5) were prepared as probes for the study of the nature of the active-site residues in the catalytic subunit of cyclic AMP dependent protein kinase. Under conditions of inhibitor in excess, both peptides inactivated the catalytic subunit by an apparent biphasic process. A fast phase, which inactivated the protein by approximately 40%, was followed by a slow phase that accounted for the loss of the remaining enzyme activity. Protection experiments with the kinase substrates showed that the slow phase of inactivation was active site directed, while the fast phase was not. Studies with radioactively labeled peptides 4 and 5 indicated incorporation of two peptide residues per molecule of the catalytic subunit upon complete inactivation. This observation is consistent with the occurrence of one alkylation event in each phase of the inactivation. The protein was proteolyzed subsequent to its modification with radioactive peptides. High-performance liquid chromatography afforded two radioactive peptide fragments in each case, which were sequenced by Edman degradation. Peptide 4 alkylated Thr-197 and Glu-346, while peptide 5 modified Cys-199 and also Glu-346. Data are presented to support the conclusion that Thr-197 and Cys-199 are located at or near the active site.
Mobashery
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Laboratory of Bioorganic Chemistry and Biochemistry, Rockefeller University, New York, New York 10021-6399.
Kaiser
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D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D007202:Indicators and Reagents; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D000345:Affinity Labels; D007202:Indicators and Reagents; D009842:Oligopeptides; D011494:Protein Kinases