Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors.

The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H(+)-PPase) from oat (Avena sativa L.) roots to changes in Mg(2+) and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H(+)-PPase is Mg(2)PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg(2+) and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H(+)-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H(+)-PPase. It is demonstrated that models incorporating Mg(2)PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H(+)-PPase. Calculations of the likely concentrations of Mg(2)PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H(+)-PPase would be about 70% saturated in vivo.