Oligomeric Functional Form of the Gastric H,K‐ATPase

Abstract

The P2 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 32P-ATP, 32Pi, binding of γ 32P-ATP and a K+-competitive inhibitor, INT. At a low concentration of MgATP (<10μM), the enzyme forms E1[ATP]·Mg·(H+):E2·Mg·(H+) at a high ATP affinity site, followed by phosphorylation to form E1P·Mg·(H+):E2·Mg·(H+), that converts to E2P·Mg·(H+):E1·Mg·(H+). Maximal protein phosphorylation was 2.65 nmol/mg enzyme. At high concentration of MgATP (>0.1 mM) the oligomer forms E1P·Mg·(H+):E2[ATP] ·Mg·(H+)which converts to E2P·Mg·(H+):E1[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 32Pi in the presence of Mg2+. 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 E2 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 E2· Mg·INTexo:E1P·Mg·(H+)cyto. Presumably the binding of the inhibitor restricts membrane domain movement, fixing its conformation in the E2 form resulting in the inhibition of enzyme activity and the other half of the oligomer must remain in E1P form. The combined data suggest that the oligomer is present always in a functional E1E2 configuration during enzyme turnover.