Hagiwara M, Inagaki M, Hidaka H
Mol Pharmacol. 1987 May;31(5):523-8.
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.
研究了牛心肌cAMP依赖性蛋白激酶催化亚基与N-[2-(甲氨基)乙基]-5-异喹啉磺酰胺（H-8）的相互作用，H-8是异喹啉磺酰胺衍生物系列中对环核苷酸依赖性蛋白激酶最有效且最具选择性的抑制剂。添加H-8以剂量依赖的方式保护酶的催化亚基免受ATP类似物对氟磺酰苯甲酰-5'-腺苷（FSBA）的不可逆失活作用。失活遵循假一级动力学，H-8降低了失活的稳态常数（Ki），而对一级速率常数（K3）没有任何影响。在热力学平衡条件下，使用凝胶过滤法测量H-8与酶的定量结合。催化亚基与蛋白质的结合量约为1摩尔药物/摩尔蛋白质，在药物浓度为1.0 microM时达到表观半最大结合，而被FSBA不可逆修饰的酶不结合该药物，这证实了除活性位点外，酶的催化亚基中没有H-8的结合位点。结合研究还表明，H-8不需要Mg2+等二价阳离子来结合蛋白激酶的催化亚基。使用FSBA和其他推测可修饰催化亚基活性位点或其附近残基的亲和标记试剂来表征H-8与活性位点的结合。H-8保护酶免受FSBA和Cibacron Blue F3GA的失活作用，但对5,5'-二硫代双-(2-硝基苯甲酸)（DTNB）和7-氯-4-硝基-2,1,3-苯并恶二唑（NBD-Cl）的共价修饰没有提供任何保护，这表明H-8的结合位点不涉及催化亚基中ATP结合位点的γ亚位点，因为DTNB和NBD-Cl被认为可修饰与ATP分子γ磷酸互补的残基。
