Modification of histidine 5 in sarcoplasmic reticulum Ca2+-ATPase by diethyl pyrocarbonate causes strong inhibition of formation of the phosphoenzyme intermediate from inorganic phosphate.

Sarcoplasmic reticulum vesicles were modified with diethyl pyrocarbonate (DEPC), a histidine-modifying reagent. Phosphoenzyme formation from Pi in the Ca2+-ATPase (reversal of hydrolysis of the phosphoenzyme intermediate) was almost completely inhibited by this modification. Tight binding of F- and Mg2+ and high affinity binding of vanadate in the presence of Mg2+, both of which produce transition state analogs for phosphoenzyme formation from the magnesium-enzyme-phosphate complex, were also inhibited. Formation of the phosphoenzyme from acetyl phosphate in the forward reaction was only weakly inhibited, but hydrolysis of the phosphoenzyme was strongly inhibited. The enzyme was protected by tight binding of F- and Mg2+ or by high affinity binding of vanadate in the presence of Mg2+ against the DEPC-induced inhibition of phosphoenzyme formation from Pi. The enzyme was also protected by tight binding of F- and Mg2+ against the DEPC-induced inhibition of phosphoenzyme hydrolysis. Peptide mapping of the tryptic digests, detection of peptides containing DEPC-modified histidine by UV absorption at 240 nm, amino acid analysis, sequencing, and mass spectrometry showed that His-5 was a single major residue protected by the above transition state analogs against the modification with DEPC. These results indicate that modification of His-5 with DEPC is responsible for the DEPC-induced inhibition of phosphoenzyme formation from Pi and of phosphoenzyme hydrolysis and suggest that His-5 is located in or very close to the catalytic site in the transition state for phosphoenzyme formation from the magnesium-enzyme-phosphate complex and is likely involved in the catalytic process of this reaction step.