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 [mu]M. Protection from inhibition by N-ethylmaleimide and phenylglyoxal varied as a function of Mg2PPi concentration, suggesting that this is the substrate for the H+-PPase. Protection by Mg2PPi followed Michaelis-Menten kinetics with a Km of approximately 2 [mu]M. 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 [mu]M) and Mg2PPi as the substrate (Km = 2.5-5 [mu]M).
Gordon-Weeks
R.
R
Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, United Kingdom.
Steele
S. H.
SH
Leigh
R. A.
RA