The Role of Magnesium, Pyrophosphate, and Their Complexes as Substrates and Activators of the Vacuolar H⁺-Pumping Inorganic Pyrophosphatase: Studies Using Ligand Protection from Covalent Inhibitors

Inhibitors preferentially and covalently reactive with cysteine, arginine, histidine, and carboxyl-containing residues were inhibitory to the plant vacuolar H+-transporting inorganic pyrophosphatase (H+-PPase) from Vigna radiata (mung bean) and Beta vulgaris (red beet), but hydrophobic compounds and those reactive with tyrosine and lysine were less effective. Inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, phenylglyoxal, and N-ethylmaleimide was decreased in the presence of Mg2+ or mixtures of Mg2+ and inorganic pyrophosphate (PPi) but not by PPi alone. None of these ligands affected inhibition by reagents reactive with histidine. The Mg2+ dependence of protection from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide inhibition followed first-order kinetics and yielded a Km for free Mg2+ of 20 to 23 μM. Protection from inhibition by N-ethylmaleimide and phenylglyoxal varied as a function of $\text{Mg}_{2}\text{PPi}$ concentration, suggesting that this is the substrate for the H+-PPase. Protection by $\text{Mg}_{2}\text{PPi}$ followed Michaelis-Menten kinetics with a Km of approximately 2 μM. These results are consistent with the predictions of a kinetic model for the H+-PPase (R. A. Leigh, A. J. Pope, I. R. Jennings, D. Sanders [1992] Plant Physiol 100: 1698-1750), which identified free Mg2+ as an allosteric activator (Km = 25 μM) and $\text{Mg}_{2}\text{PPi}$ as the substrate (Km = 2.5-5 μM).