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

Abstract

The maximum amount of acid-stable phosphoenzyme (E³²P)/mol of α chain of pig gastric H/K-ATPase from [γ-³²P]ATP (K_1/2 = 0.5 μM) was found to be ∼0.5, which was half of that formed from ³²P_i (K_1/2 = 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 K_1/2 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 P_i liberation, both of which decreased with increasing concentrations of ATP. The ratio of the amount of P_i 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 P_i 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⁺.