Adenosine 3',5'-cyclic monophosphate protein kinase from bovine brain: inactivation of the catalytic subunit and holoenzyme by 7-chloro-4-nitro-2,1,3-benzoxadiazole.

NBD-C1 (7-chloro-4-nitro-2,1,3-benzoxadiazole) inactivates the catalytic subunit of adenosine 3’,5’-cyclic monophosphate (CAMP) depthdent protein kinase isolated from bovine brain by covalent modification. The reaction follows pseudo-first-order kinetics. The pseudo-first-order rate constant (k8p,,) shows a hyperbolic dependence on NBD-Cl concentration, suggesting formation of a reversible complex before covalent modification with a dissociation constant (4) of 150 pM. MgATP, MgADP, and adenosine protect against inactivation 75,60, and SO%, respectively, whereas ATP, ADP, or Mg2+ alone does not protect. This process was competitive in nature ( K , for adenosine 44 pM). Protein substrates (histone IIa, 2 mg/mL; Leu-Arg-Arg-Ala-ser-Leu-Gly, 1 mM) do not protect against inactivation; histone actually accelerates the rate of inactivation. Inactivation is associated with modification of 2.1 f 0.15 mol of cysteine/mol of catalytic subunit, determined spectrophotometrically and radioisotopically. Activity can be restored by treating inactivated enzyme with 2-mercaptoethanol. MgATP protects one cysteine on the Adenos ine 3’,5’-cyclic monophosphate (CAMP) dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) catalyzes the phosphorylation of polypeptidic serine and threonine residues. The enzyme consists of dissimilar regulatory (R) and catalytic (C) subunits.’ Until recently the enzyme was reported to be activated by binding 2 mol of cAMP and dissociating into free C subunits and dimeric R2(cAMP)2 complexes (Gill & Garen, 1970; Tao et al., 1970; Rosen & Erlichman, 1975; Beavo et al., 1975). Recent studies, however, indicate that each regulatory dimer can bind 4 mol of cAMP (Corbin et al., 1978; Weber et al., 1979), yielding the following activation scheme: R2C2 + 4cAMP + R~(cAMP), + 2C From the Department of Biochemistry, Louisiana State University Medical Center, New Orleans, Louisiana 701 12. Received January 14, 1981; revised manuscript received April 14, 1982. This work was supported by Grant NS-15994 from the U S . Public Health Service. 0006-2960/82/0421-5 175$01.25/0 average from modification while protecting against inactivation. In the absence of CAMP, type I and I1 regulatory subunits from bovine skeletal muscle and type I1 regulatory subunit from bovine brain slow modification of the catalytic subunit by NBD-Cl. In the presence of 0.6 mM cAMP the brain holoenzyme is inactivated slower than the free catalytic subunit, but type I and I1 holoenzymes from skeletal muscle are inactivated faster than the free catalytic subunit. This suggests that the regulatory and catalytic subunits communicate even in the presence of saturating concentrations of CAMP. The effect of brain regulatory subunit on the reactivity of sulfhydryls in the catalytic subunit is opposite that of the skeletal muscle regulatory subunits. The latter’s effect on sulfhydryl reactivity is similar to that observed by Armstrong and Kaiser for the bovine heart enzyme [Armstrong, R. N., & Kaiser, E. T. (1978) Biochemistry 17, 28401, suggesting differences in the interactions of the regulatory and catalytic subunits in the enzymes from these different tissues. Work in this and other laboratories is aimed at identifying amino acids present at the active site of the C subunit as well as studying the mechanism of inactivation of the C subunit by regulatory proteins. Possible amino acid residues important to enzyme function have been assessed by chemical modification studies. Characterization of ethoxyformic anhydride inhibition of C subunit (Witt & Roskoski, 1975a), for example, suggests that a tyrosine residue occurs in the active site. Chemical modification of cysteine residues also inhibits enzyme activity (Sugden et al., 1976; Peters et al., 1977; Armstrong & Kaiser, 1978). I Abbreviations: R, regulatory subunit of CAMP-dependent protein kinase; C, catalytic subunit of CAMP-dependent protein kinase; FSBA, 5’-[p-(fluorosulfonyl)benzoyl]adenosine; NBD-CI, 7-chloro-4-nitro2,1,3-benzoxadiazole; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid); Mops, 3-(N-morpholino)propanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; Ser-peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly; DEAE, diethylaminoethyl; NaDodS04, sodium dodecyl sulfate.