Oligomeric Functional Form of the Gastric H,K‐ATPase

The P 2 type gastric H,K‐ATPase undergoes a cycle of conformational changes involving phosphorylation and dephsophorylation during H + for K + exchange. The functional consequences of the dimeric structure was investigated by phosphorylation with γ 32 P‐ATP, 32 Pi, binding of γ 32 P‐ATP and a K + ‐competitive inhibitor, INT. At a low concentration of MgATP (<10μM), the enzyme forms E 1 [ATP]·Mg·(H+):E 2 ·Mg·(H+) at a high ATP affinity site, followed by phosphorylation to form E 1 P·Mg·(H+):E 2 ·Mg·(H + ), that converts to E 2 P·Mg·(H + ):E 1 ·Mg·(H + ). Maximal protein phosphorylation was 2.65 nmol/mg enzyme. At high concentration of MgATP (>0.1 mM) the oligomer forms E 1 P·Mg·(H + ):E 2 [ATP] ·Mg·(H + )which converts to E 2 P·Mg·(H + ):E 1 [ATP] ·Mg·(H + ). The combination of maximal phosphorylation and ATP binding was 5.2 nmol/mg enzyme, similar to the value found for phosphorylation from 32 Pi in the presence of Mg 2+ . INT inhibited the gastric H,K‐ATPase K + competitively. Maximal binding of INT was 2.64 nmole per mg of the enzyme in the presence of MgATP. K + ion displaced INT bound in intact vesicles only in the presence of nigericin showing that INT binding is only to the luminal E 2 form. INT‐bound enzyme also formed 2.63 nmole of EP per mg protein, which is insensitive to K + . Hence a total of 5.29 nmol binding sites are present per mg of enzyme, with equal binding stoichiometry of INT and phosphoenzyme. INT binding results in the formation of E 2 · Mg·INT exo :E 1 P·Mg·(H + ) cyto . Presumably the binding of the inhibitor restricts membrane domain movement, fixing its conformation in the E 2 form resulting in the inhibition of enzyme activity and the other half of the oligomer must remain in E 1 P form. The combined data suggest that the oligomer is present always in a functional E 1 E 2 configuration during enzyme turnover.