Modification of arginine-198 in sarcoplasmic reticulum Ca2+-ATPase by 1,2-cyclohexanedione causes inhibition of formation of the phosphoenzyme intermediate from inorganic phosphate.
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.
Saino
T
T
Department of Biochemistry, Asahikawa Medical College, Nishikagura Asahikawa 078, Japan.
Daiho
T
T
Kanazawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D003512:Cyclohexanones / Q000737:chemistry*; D005459:Fluorides / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014638:Vanadates / Q000737:chemistry
D003512:Cyclohexanones; D010710:Phosphates; D014638:Vanadates; C018031:1,2-cyclohexanedione; D001120:Arginine; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D005459:Fluorides