8-Azido-2'-O-dansyl-ATP. A fluorescent photoaffinity reagent for ATP-binding proteins and its application to adenylate kinase.
The photoaffinity reagent 8-azido-2'-O-[14C]dansyl-ATP (AD-ATP) has been synthesized for labeling and monitoring the active sites of ATPases and kinases. In its first application, the reagent is used to explore the active site of adenylate kinase from rabbit muscle. In the dark, AD-ATP inhibits adenylate kinase reversibly and competitively with KI = 0.25 +/- 0.01 microM. Under weak UV illumination, AD-ATP labels adenylate kinase irreversibly. The photoinactivation data also show KI = 0.25 +/- 0.02 microM. The ratio (r) of the specific activity of AD-ATP-labeled adenylate kinase to that of the unlabeled enzyme has been determined as a function of the number (n) of label/enzyme. The linear plot of r versus n with slope equal to -1 shows that the labeling is very specific, i.e. each label completely inactivates an enzyme molecule. After the labeled enzyme was partially hydrolyzed and the radioactive peptides analyzed and sequenced, it was found that Leu-115, Cys-25, and probably His-36 were labeled, in agreement with previous conclusions on the structure of the active site of this enzyme based on amino acid sequence, x-ray diffraction, and NMR studies. The environment-sensitive fluorescent dansyl group of AD-ATP can function as an in situ probe for monitoring ligand or conformation changes at the active site. The fluorescence of AD-ATP-labeled enzyme with n = 0.9 is not affected by ATP but increases with the concentration of AMP in solution. This observation is also in agreement with the previous conclusion that ATP does not bind to the AMP site of adenylate kinase. The observed enhancement of fluorescence indicates that binding of AMP by this enzyme causes environmental change at its ATP site. The possible usefulness of AD-ATP as an effective biological inhibitor or as a molecular probe for studying the structure and regulation of ATP-binding proteins is discussed.
Chuan
H
H
Bioenergetics Laboratory, State University of New York, Buffalo 14214-3094.
Lin
J
J
Wang
J H
JH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000263:Adenylate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001386:Azides / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D003619:Dansyl Compounds / Q000378:metabolism* / Q000494:pharmacology; D005456:Fluorescent Dyes; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000378:metabolism*; D010777:Photochemistry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014961:X-Ray Diffraction
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; C059710:8-azido-2'-O-dansyladenosine triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000263:Adenylate Kinase