title: Specific binding of a novel compound, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) to the active site of cAMP-dependent protein kinase.
abstract: The interaction of the catalytic subunit of bovine cardiac muscle cAMP-dependent protein kinase with N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the most potent and selective inhibitor toward cyclic nucleotide-dependent protein kinases in the series of isoquinolinesulfonamide derivatives, was studied. The addition of H-8 protected the catalytic subunit of the enzyme in a dose-dependent manner from irreversible inactivation by the ATP analogue p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). The inactivation followed pseudo-first order kinetics and H-8 reduced the steady state constant of inactivation (Ki) without any effect on the first order rate constant (K3). The quantitative binding of H-8 to the enzyme was measured under conditions of thermodynamic equilibrium using a gel filtration method. The catalytic subunit bound approximately 1 mol of drug/mol of protein with apparent half-maximal binding at 1.0 microM drug, whereas the enzyme irreversibly modified by FSBA did not bind the drug, confirming that the enzyme has no site for H-8 in the catalytic subunit other than the active site. The binding studies also showed that H-8 does not require divalent cations such as Mg2+ to bind to the catalytic subunit of the protein kinase. The binding of H-8 to the active site was characterized using FSBA and other affinity labeling reagents which have been postulated to modify residues at or near the active site of the catalytic subunit. H-8 protected the enzyme against inactivation by FSBA and Cibacron Blue F3GA but did not afford any protection against the covalent modification of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), suggesting that the binding site of H-8 does not involve the gamma-subsite of the ATP binding site in the catalytic subunit, since DTNB and NBD-Cl are thought to modify the residues complementary to gamma-phosphate of the ATP molecules.
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000098:Acetophenones / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D047428:Protein Kinase Inhibitors; D013329:Structure-Activity Relationship; D045162:Thiazolidinediones; D014227:Triazines / Q000494:pharmacology; D009764:o-Phthalaldehyde / Q000494:pharmacology
chemical_list: D000098:Acetophenones; D000345:Affinity Labels; D002364:Caseins; D006657:Histones; D047428:Protein Kinase Inhibitors; D045162:Thiazolidinediones; D014227:Triazines; C001995:5-carboxymethyl-3-p-tolylthiazolidine-2,4-dione-2-acetophenone; C012842:Cibacron Blue F 3GA; D009764:o-Phthalaldehyde; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D009327:4-Chloro-7-nitrobenzofurazan; D008274:Magnesium; D000241:Adenosine