3'-O-(5-fluoro-2,4-dinitrophenyl)-ATP exclusively labels Lys-492 at the active site of the sarcoplasmic reticulum Ca(2+)-ATPase.
Sarcoplasmic reticulum vesicles were labeled with 40 microM 3'-O-(5-fluoro-2,4-dinitrophenyl)-ATP (FDNP-ATP) at 25 degrees C and pH 7.0 for 4 h. The Ca(2+)-ATPase was inhibited strongly. The enzyme was almost completely protected either by 20 mM Mg.ATP or by 50 mM acetyl phosphate against this inhibition. Pi gave no protection. There was a linear relationship between the extent of this inhibition and the Mg.ATP-sensitive part of the content of bound FDNP-ATP. Extrapolation showed that the enzyme is completely inhibited by Mg.ATP-sensitive binding of 3.6 nmol of FDNP-ATP/mg of the vesicle protein. This value is in good agreement with the content of the phosphorylation site (3.3 nmol/mg of the vesicle protein) in the vesicles used. These findings indicate that binding of 1 mol of FDNP-ATP per mol of the active sites leads to a complete inhibition of the enzyme. The acetylphosphatase activity and phosphorylation with ATP were also strongly inhibited by this labeling, whereas phosphorylation with Pi was not inhibited. The labeled vesicles were solubilized in SDS, and the Ca(2+)-ATPase was purified by size exclusion high performance liquid chromatography. Mapping the labeled peptides in the tryptic digest by reversed-phase high performance liquid chromatography and sequencing showed that Lys-492 was exclusively labeled with FDNP-ATP. These results show that Lys-492 is located in or near the ATP binding site and apart from the phosphorylation site and Pi binding site. Molecular modeling of FDNP-ATP suggests that this Lys-492 residue is situated on the 3'-OH side of the ribose moiety of bound ATP and is close to the alpha-phosphoryl group.
Yamasaki
K
K
Department of Biochemistry, Asahikawa Medical College, Japan.
Daiho
T
T
Kanazawa
T
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000345:Affinity Labels; D010446:Peptide Fragments; C057111:3'-O-(5-fluoro-2,4-dinitrophenyl)ATP ether; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008239:Lysine