Identification of arginyl residues located at the ATP binding site of sarcoplasmic reticulum Ca2+-ATPase. Modification with 1,2-cyclohexanedione.
Sarcoplasmic reticulum vesicles were treated with 1, 2-cyclohexanedione (CHD) in sodium borate (pH 8.0). The Ca2+-ATPase activity was completely inhibited. Inhibition of Mg.ATP and Mg.ADP binding to the high affinity ATP binding site as well as inhibition of phosphorylation with ATP occurred simultaneously with the inhibition of the Ca2+-ATPase activity. Phosphorylation with acetyl phosphate was not inhibited. The Ca2+-ATPase was strongly protected by Mg.ATP, Mg.ADP, and Mg.AMP against this inhibition. Binding of acetyl phosphate or Pi to the enzyme gave no protection. Phosphorylation with acetyl phosphate also had no protective effect. Peptide mapping of the tryptic digests, detection of peptides containing CHD-modified arginyl residues with Girard's reagent T, and sequencing revealed that Arg-489, Arg-505, and Arg-678 were modified with CHD. Arg-489 and Arg-678 were almost completely protected by Mg.ATP against this modification, but partially protected by prelabeling with fluorescein 5-isothiocyanate, which occupies the adenosine binding region in the ATP binding site. In contrast, Arg-505 was slightly protected by Mg.ATP and almost completely protected by prelabeling with fluorescein 5-isothiocyanate. Taken together, these findings suggest that Arg-489 and Arg-678 are located in or near the region occupied by the triphosphate moiety of ATP, either or both of these residues being in or close to the region occupied by the alpha-phosphoryl group in the high affinity ATP binding site and involved in the CHD-induced inhibition of this enzyme and that Arg-505 is very close to (but slightly out of) the adenosine binding region in the ATP binding site. The acetyl phosphatase activity and phosphorylation with Pi were also inhibited by the CHD treatment, but the inhibitions were considerably slower than those described above. This suggests that the arginyl residues involved in these inhibitions are distinct from that involved in the inhibition of the Ca2+-ATPase activity.
Kimura
K
K
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan. kanazawa@asahikawa-med.ac.jp
Suzuki
H
H
Daiho
T
T
Yamasaki
K
K
Kanazawa
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003512:Cyclohexanones / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D003512:Cyclohexanones; D004791:Enzyme Inhibitors; C018031:1,2-cyclohexanedione; D000255:Adenosine Triphosphate; D001120:Arginine; D014357:Trypsin; D000252:Calcium-Transporting ATPases