Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase.
The yeast plasma membrane proton-pumping ATPase forms a phosphorylated intermediate during the hydrolysis of ATP. The fraction of enzyme phosphorylated during steady-state ATP hydrolysis was studied as a function of substrate concentration (MgATP), Mg2+ concentration, and pH. The dependence of the fraction of enzyme phosphorylated on the concentration of MgATP is sigmoidal, and the isotherms can be fit with parameters and mechanisms similar to those used to describe ATP hydrolysis. The isotherm is significantly more sigmoidal at pH 5.5 than at pH 6.0, with the limiting percentage (100.mol of phosphate/mol of enzyme) of enzyme phosphorylated being 70% and 6%, respectively, at the two pH values. The maxima in the steady-state rate of ATP hydrolysis occur at higher concentrations of Mg2+ and higher pH than the maxima in the fraction of enzyme phosphorylated. This suggests that the rate-determining step for ATP hydrolysis is different from that for enzyme phosphorylation and the hydrolysis of phosphoenzyme is enhanced by Mg2+ and high pH. The rate of phosphoenzyme formation was investigated with the quenched-flow method, but only a lower bound of 140 s-1 could be obtained for the rate constant at MgATP concentrations greater than 2.5 mM. Since the turnover number for ATP hydrolysis under similar conditions is 14 s-1, the rate-determining step in ATP hydrolysis occurs after enzyme phosphorylation.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014638:Vanadates / Q000494:pharmacology
D010750:Phosphoproteins; D014638:Vanadates; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium