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 K-m for free Mg2+ Of 20 of 23 mu M Protection from inhibition by N-ethylmaleimide and phenylglyoxal varied as a function of Mg(2)PPi concentration, suggesting that this is the substrate for the H+-PPase. Protection by Mg(2)PPi followed Michaelis-Menten kinetics with a K-m of approximately 2 mu M. These results are consistent with the predictions of a kinetic model for the H+-PPase (R.A. Leigh, A.). Pope, I.R. Jennings, D. Sanders [1992] Plant Physiol 100: 1698-1750), which identified free Mg2+ as an allosteric activator (K-m = 25 mu M) and Mg(2)PPi as the substrate (K-m = 2.5-5 mu M).