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+(K50approximately 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 Mg2+for activation. At saturating concentrations of free Mg2+, H+-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi2-but not Mg2PPi, demonstrating that MgPPi2-was the true substrate of the enzyme. The apparent Km(MgPPi2-) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H+transport (7 micromolar). Free Mg2+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 Mg2+concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg2+. In contrast, cytosolic concentrations of K+and MgPPi2-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 Mg2+would be released from the MgATP2-complex, thereby activating the tonoplast H+-PPiase.