Adenosine cyclic 3',5'-monophosphate dependent protein kinase: active site directed inhibition by Cibacron Blue F3GA

Publisher Website
Google Scholar
Abstract
Cibacron Blue F3GA, the polycyclic blue chromophore of Blue Dextran 2000, inhibits the catalytic subunit of bovine brain protein kinase. The rate of inactivation exhibits a hyperbolic dependence on the dye concentration. An enzyme-dye complex probably forms prior to inactivation. Protein and peptide substrates or MgATP protects the enzyme against dye inactivation. Kinetic measurements show that the dissociation constant is .apprx. 100 .mu.M and the maximal rate of inactivation is 0.13 min-1 at 22.degree. C. Inactivation is temperature- and time-dependent. Exhaustive dialysis, gel filtration, or the addition of substrate fails to reactivate inhibited enzyme. The failure to reverse the inhibition suggests that the dye forms a covalent complex with the enzyme. Denaturation by sodium dodecyl sulfate also fails to dissociate the dye from enzyme. The hyperbolic kinetics suggest that the dye functions as an active site directed reagent. The holoenzyme is resistant to Cibacron Blue inactivation. Addition of cycli cAMP converts the enzyme to a form susceptible to inhibition. The regulatory subunit probably shields, either physically or functionally, the active site of the catalytic subunit.