Steady state kinetic studies of purified yeast plasma membrane proton-translocating ATPase.

The plasma membrane H+-ATPase from bakers' yeast was purified and reconstituted with phosphatidylserine. The steady state kinetics of ATP hydrolysis catalyzed by the H+-ATPase were studied over a wide range of Mg2+ and ATP concentrations. Whereas MgATP was the substrate hydrolyzed, excess concentrations of either Mg2+ or ATP were inhibitory. The dependence of the steady state initial velocity of ATP hydrolysis on the concentration of MgATP at a fixed concentration of Mg2+ was sigmoidal rather than hyperbolic. This precluded mechanisms involving only activation and inhibition by Mg2+ and competitive inhibition by ATP. Two alternative interpretations of these results are: 1) the enzyme possesses multiple catalytic sites which interact cooperatively; or 2) the enzyme can exist in multiple conformational states which catalyze MgATP hydrolysis by parallel pathways. The rate laws for both mechanisms are identical so that the two mechanisms cannot be distinguished on the basis of the kinetic data. The data are well fit by the rate law for these mechanisms with the inclusion of competitive inhibition by Mg2+ and ATP and an independent inhibition site for Mg2+.