Modification of arginine-198 in sarcoplasmic reticulum Ca2+-ATPase by 1,2-cyclohexanedione causes inhibition of formation of the phosphoenzyme intermediate from inorganic phosphate.

T Saino, T Daiho, T Kanazawa

文献索引：J. Biol. Chem. 272(34) , 21142-50, (1997)

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摘要

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.