Gastric H/K-ATPase Liberates Two Moles of Pi from One Mole of Phosphoenzyme Formed from a High-Affinity ATP Binding Site and One Mole of Enzyme-Bound ATP at the Low-Affinity Site during Cross-Talk between Catalytic Subunits

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Abstract
The maximum amount of acid-stable phosphoenzyme (E32P)/mol of α chain of pig gastric H/K-ATPase from [γ-32P]ATP (K1/2 = 0.5 μM) was found to be ∼0.5, which was half of that formed from 32Pi (K1/2 = 0.22 mM). The maximum 32P binding for the enzyme during turnover in the presence of [γ-32P]ATP or [α-32P]ATP was due to 0.5 mol of E32P + 0.5 mol of an acid-labile enzyme-bound [γ-32P]ATP (EATP) or 0.5 mol of an acid-labile enzyme-bound [α-32P]ATP, respectively. The K1/2 for EATP formation in both cases was 0.12 ∼ 0.14 mM. The turnover number of the enzyme (i.e., the H+-ATPase activity/(EP + EATP)) was very close to the apparent rate constants for EP breakdown and Pi liberation, both of which decreased with increasing concentrations of ATP. The ratio of the amount of Pi liberated to that of EP that disappeared increased from 1 to ∼2 with increasing concentrations of ATP (i.e., equal amounts of EP and EATP exist, both of which release phosphate in the presence of high concentrations of ATP). This represents the first direct evidence, for the case of a P-type ATPase, in which 2 mol of Pi liberation occurs simultaneously from 1 mol of EP for half of the enzyme molecules and 1 mol of EATP for the other half during ATP hydrolysis. Each catalytic α chain is involved in cross-talk, thus maintaining half-site phosphorylation and half-site ATP binding which are induced by high- and low-affinity ATP binding, respectively, in the presence of Mg2+.