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.