Substrate kinetics of the tonoplast H<sup>+</sup>-translocating inorganic pyrophosphatase and its activation by Free Mg<sup>2+</sup>
White PJ., Marshall J., Smith JAC.
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.