A potent fluorescent ATP-like inhibitor of cAMP-dependent protein kinase.
The fluorescent ATP analogue 8-azido-2'-O-[14C]dansyl-ATP ([ 14C]AD-ATP) was used to probe the ATP-binding site in the catalytic (C) subunit of cAMP-dependent protein kinase. AD-ATP was found to inhibit the phosphotransferase activity of C subunit with extremely high specificity. Complete inhibition was observed when each mol of C subunit was covalently labeled with 1 mol of this fluorescent ATP analogue. The labeling can be accelerated by the presence of Mg2+ or Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), whereas high concentrations of ATP can almost completely protect the enzyme from AD-ATP. Detailed studies indicated that AD-ATP competes with ATP for binding to C subunit. Analysis of the kinetic data gave dissociation constants of 2.9 and 13 microM for AD-ATP and ATP bound to C subunit, respectively. AD-ATP has a fluorescence emission peak at 510 nm in pH 7.0 aqueous buffer containing 25% glycerol. After covalent binding to C subunit this emission peak shifts to 455 nm, which suggests that the label at ATP site is in an endogenous hydrophobic environment. Upon the binding of Mg2+ or Kemptide, the fluorescence of AD-ATP-labeled C subunit can be enhanced by 50 and 45%, respectively. This enhancement suggests that the binding of either the peptide substrate or Mg2+ induces conformational change at the active site of C subunit. Analysis of the fluorescence data shows that the values of Kd for Mg2+ and Kemptide bound to AD-ATP-labeled C subunit are 0.2 mM and 2.1 microM, respectively. The normal procedure for the preparation of the C subunit from the bovine heart muscle has been simplified to require only one-fifth of the usual working time to obtain the homogeneous enzyme with 70% yield from the crude extract.
Wu
J C
JC
Bioenergetics Laboratory, State University of New York, Buffalo 14214-3094.
Chuan
H
H
Wang
J H
JH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology*; D003619:Dansyl Compounds / Q000378:metabolism / Q000494:pharmacology*; D005456:Fluorescent Dyes; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology; D009842:Oligopeptides / Q000494:pharmacology; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000345:Affinity Labels; D001386:Azides; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; C059710:8-azido-2'-O-dansyladenosine triphosphate; C037642:kemptide; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D008274:Magnesium