Steady-state kinetics of the plasma membrane H+-ATPase from red beet (Beta vulgaris): its inhibition by vanadate and inorganic phosphate

Abstract

The intial velocity vs ATP concentration curves obtained with the plasma membrane H+-ATPase from red beet (Beta vulgaris L.) did not follow classical Michaelis-Menten kinetics. A rate equation containing second-order terms in ATP concentration in both the numerator and the denominator was used to obtain a significantly better fit to the data. The observed deviations from Michaelis-Menten kinetics were more pronounced in the presence of potassium ions. The inhibition caused by inorganic phosphate was partial. i.e. the ATPase activity extrapolated at an infinite phosphate concentration was not zero. In contrast, the inhibition produced by orthovanadate was nearly total. The inhibitions caused by both phosphate and vanadate were uncompetitive with respect to ATP and enhanced by potassium ions and high concentrations of dimethyl sulfoxide. a solvent used to lower the water activity of the reaction medium. The ATP-dependent proton transport was stimulated by potassium ions and was inhibited by phosphate only at high ATP concentrations. A kinetic mechanism, in which the H+-ATPase can adopt two conformations during its catalytic cycle and can form a ternary enzyme-ATP-phosphate complex able to hydrolyze bound ATP. is proposed to explain those results.