Evidence from 18O exchange measurements for steps involving a weak acid and a slow chemical transformation in the mechanism of phosphorylation of the gastric H+, K+-ATPase by inorganic phosphate.
Phosphorylation of the gastric H,K-ATPase by Pi has been studied by measuring the P18Oj16O4-j distribution as a function of time at different H+, K+, and [18O]Pi concentrations. The advantage of isotope exchange measurements is that the P18Oj16O4-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 (v(ex] 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.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 occurring by a different mechanism when a group with pKa = 7.2 is protonated. Slower than diffusion-controlled formation of the E.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.
Faller
L D
LD
Department of Medicine, UCLA School of Medicine.
Diaz
R A
RA
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D017506:H(+)-K(+)-Exchanging ATPase; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008861:Microsomes / Q000201:enzymology; D008962:Models, Theoretical; D009130:Muscle, Smooth / Q000201:enzymology; D010103:Oxygen Isotopes; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013270:Stomach / Q000201:enzymology; D013552:Swine
D010103:Oxygen Isotopes; D010710:Phosphates; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase