Adenosine cyclic 3',5'-monophosphate-dependent protein kinase: Interaction of the catalytic subunit and holoenzyme with lin-benzoadenine nucleotides

F. Thomas Hartl, Robert Roskoski, Mary S. Rosendahl, Nelson J. Leonard
1983 Biochemistry  
The interaction of lin-benzoadenosine di-and triphosphates with the catalytic subunit and type I1 holoenzymes of adenosine cyclic 3',5'-monophosphate (CAMP) dependent protein kinase has been investigated by steady-state kinetics and fluorescence spectroscopy. lin-Benzo-ADP is a competitive inhibitor of the catalytic subunit with respect to ATP with a Ki (8.0 pM) similar to the Ki for ADP (9.0 pM). This value agrees well with the Kd (9.0 pM) determined by fluorescence polarization titration.
more » ... I1 holoenzymes from bovine brain and skeletal muscle have Kd values for linbenzo-ADP of 3.4 pM and 3.5 pM, respectively, and each binds approximately 2 mol/mol of R2C2 tetramer. Furthermore, fluorescence polarization studies indicate that both the catalytic subunit and type I1 holoenzyme bind lin-benzo-ADP rigidly, so that there is little or no rotation of the lin-benzo-A d e n o s i n e cyclic 3',5'-monophosphate dependent protein kinase (ATP:protein phosphotransferase; EC catalyzes the phosphorylation of serine and threonine residues in proteins using MgATP as phosphoryl donor. The enzyme exists in the cell as an inactive tetramer composed of dissimilar regulatory (R) and catalytic (C) subunits and is activated by CAMP' as follows (Corbin et al., 1978) : Abbreviations: CAMP, adenosine cyclic 3',5'-monophosphate; Mops, 3-(N-morpholino)propanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; Ser-peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly; dansyl-Serpeptide, Nu-dansyl-Leu-Arg-Ala-Ser-Leu-Gly; <ATP, 1 ,P-etheno-ATP HPLC, high-performance liquid chromatography; NBD-C1.7-chloro-4nitro-2,1,3-benzoxadiazole; DEAE, diethylaminoethyl; DTNB, 5,S-dithiobis(2-nitrobenzoic acid).
doi:10.1021/bi00279a007 pmid:6305401 fatcat:trrmwzkhhnfbdfifsy2jipxb5q