Substrate Kinetics of the Tonoplast H+-Translocating Inorganic Pyrophosphatase and Its Activation by Free Mg2+

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Abstract
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+ (K50 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 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.