Modification of Arginine-198 in Sarcoplasmic Reticulum Ca2+-ATPase by 1,2-Cyclohexanedione Causes Inhibition of Formation of the Phosphoenzyme Intermediate from Inorganic Phosphate

• Published in: The Journal of biological chemistry Vol. 272; no. 34; pp. 21142 - 21150
• Main Authors: Saino, Tomoyuki, Daiho, Takashi, Kanazawa, Tohru
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.08.1997; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Arginine - chemistry; Calcium-Transporting ATPases - chemistry; Cyclohexanones - chemistry; Fluorides - chemistry; Magnesium - chemistry; Molecular Sequence Data; Peptide Mapping; Phosphates - metabolism; Phosphorylation; Rabbits; Sarcoplasmic Reticulum - enzymology; Vanadates - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.34.21142

Sarcoplasmic reticulum vesicles were modified with 1,2-cyclohexanedione (CHD), a specific arginine-modifying reagent, in sodium borate (pH 8.0 or 8.8). Phosphoenzyme formation from Pi in the Ca2+-ATPase (reversal of hydrolysis of the phosphoenzyme intermediate) was almost completely inhibited by the modification with CHD. Tight binding of F− and Mg2+ and high affinity binding of vanadate in the presence of Mg2+, either of which produces a transition state analog for phosphoenzyme formation from the magnesium-enzyme-phosphate complex, were also markedly inhibited. In contrast, phosphoenzyme formation from acetyl phosphate in the forward reaction was unaffected. The enzyme was appreciably protected by tight binding of F− and Mg2+ or by high affinity binding of vanadate in the presence of Mg2+, but not by the presence of 20 mm MgCl2 alone or 150 mm Pi alone, against the CHD-induced inhibition of phosphoenzyme formation from Pi. Peptide mapping of the tryptic digests, detection of peptides containing CHD-modified arginyl residues with Girard's reagent T, sequencing, and mass spectrometry showed that Arg-198 was a single major residue protected by tight binding of F− and Mg2+ against the modification with CHD. These results indicate that modification of Arg-198 with CHD is responsible for at least a part (the portion reduced by the transition state analogs) of the CHD-induced inhibition of phosphoenzyme formation from Pi and suggest that Arg-198 is located in or close to the catalytic site in the transition state for phosphoenzyme formation from the magnesium-enzyme-phosphate complex.