The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound ( [formula omitted] from ox brain III. A minimal model

Abstract

A steady-state kinetic investigation of the effect of K + on the Na +-enzyme activity of the ( Na + + K +)- ATPase in broken membrane preparations is reported. Analysis of the kinetic patterns obtained, together with the results reported in the first two articles of this series permit the following conclusions. 1. 1. K + inhibits the Na +-enzyme (the enzyme activity measured at micromolar substrate concentrations in the presence of Na +). The inhibition is non-competitive at low and competitive at higher K + concentrations and is enhanced by free Mg 2+. 2. 2. The results indicate that the Na +-enzyme at steady-state tends to be accumulated in an enzyme-potassium complex when K + is added. 3. 3. The enzyme-potassium complex, in turn, binds Mg 2+ in a dead-end fashion. The dissociation constant for the enzyme-K-Mg complex, estimated from the data, is 7.2 mM. The same value was obtained earlier for the Mg 2+ inhibition constant of the substrate-free form of the ( Na + + K +)- enzyme (the enzyme activity measured with Na + and K + and at millimolar substrate concentrations) suggesting that the two constants describe the same equilibrium. 4. 4. On the basis of the known (optimal) activity of the ( Na + + K +)- ATPase , relative to that of the Na +-ATPase, rate constant condition is found which must be met if the Post-Albers kinetic scheme is to satisfy the data. Kinetic data for the phosphoenzyme indicate that this condition is not satisfied. 5. 5. On the basis of the kinetic results a model for the hydrolytic action of ( Na + + K +)- ATPase is proposed. This model encompasses the Post-Albers scheme