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Volume 271, Number 46, Issue of November 15, 1996 pp. 28933-28941
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of Arginyl Residues Located at the ATP Binding Site of Sarcoplasmic Reticulum Ca²⁺-ATPase
MODIFICATION WITH 1,2-CYCLOHEXANEDIONE

(Received for publication, June 5, 1996, and in revised form, July 31, 1996)

Keisuke Kimura ^§ , Hiroshi Suzuki , Takashi Daiho , Kazuo Yamasaki and Tohru Kanazawa

From the  Department of Biochemistry and the ^§ Third Department of Internal Medicine, Asahikawa Medical College, Asahikawa 078, Japan

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 P_i 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 -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 P_i 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.

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