PLANT PHYSIOLOGY , Vol 111, Issue 1 195-202, Copyright © 1996 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
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)
R. Gordon-Weeks, S. H. Steele and R. A. Leigh
Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, United Kingdom
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).
