Evidence from oxygen-18 exchange measurements for steps involving a weak acid and a slow chemical transformation in the mechanism of phosphorylation of the gastric hydrogen ion-potassium ATPase by inorganic phosphate

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Abstract
Phosphorylation of the gastric H,K-ATPase by Pi has been studied by measuring the P18Oj 16O4-j distribution as a function of time at different H+,K+, and [18O]Pi concentrations. The advantage of isotope exchange measurements is that the P18 Oj16O4-j distribution depends on the relative rates of HOH loss to form the phosphoenzyme intermediate and Pi dissociation from the enzyme. Therefore, 18O exchange is a sensitive probe of mechanism. K+ increases the exchange rate (vex) but does not affect the partition coefficient (Pc) that determines the P18Oj16O4-j distribution. Conversely, H+ inhibits exchange. A single Pc describes the data at every pH, but the value increases from 0.04 at pH 8 to 0.64 at pH 5.5. vex depends hyperbolically on [Pi]0. Km for Pi does not depend on pH, and Pc does not depend on [Pi]0. Individual rate constants in the phosphorylation mechanism are estimated. Formation of the E.cntdot.Pi complex that looses HOH is 1-2 orders of magnitude slower at pH 5.5 than at pH 8 and is not diffusion controlled. The observed change in Pc with pH is compatible with catalysis occuring by a different mechanism when a group with pKa = 7.2 is protonated. Slower than diffusion-controlled formation of the E.cntdot.Pi complex that splits out HOH is evidence for a relatively slow, unimolecular chemical transformation involving an additional intermediate in the phosphorylation mechanism, such as a protein conformational change.

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