Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg.

To clarify the kinetic characteristics and ionic requirements of the tonoplast H(+)-translocating inorganic pyrophosphatase (H(+)-PPiase), PPi hydrolysis and PPi-dependent H(+) transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H(+)-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H(+)-PPiase activity was maximal in the presence of K(+) (K(50) approximately 3 millimolar), with PPi-dependent H(+) transport being more selective for K(+) than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H(+)-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg(2+) for activation. At saturating concentrations of free Mg(2+), H(+)-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi(2-) but not Mg(2)PPi, demonstrating that MgPPi(2-) was the true substrate of the enzyme. The apparent K(m) (MgPPi(2-)) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H(+) transport (7 micromolar). Free Mg(2+) was shown to be an allosteric activator of the H(+)-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H(+) transport. Half-maximal H(+)-PPiase activity occurred at a free Mg(2+) concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg(2+). In contrast, cytosolic concentrations of K(+) and MgPPi(2-) appear to be saturating for H(+)-PPiase activity. We propose that one function of the H(+)-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H(+)-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg(2+) would be released from the MgATP(2-) complex, thereby activating the tonoplast H(+)-PPiase.