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)

Abstract

lnhibitors 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 radiafa (mung bean) and Befa vulgaris (red beet), but hydrophobic compounds and those reactive with tyrosine and lysine were less effective. lnhibition by 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide, phenylglyoxal, and N-ethylmaleimide was decreased i n the presence of Mgz+ 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-dimethylaminopropy1)carbodiimide inhibition followed first-order ki-

Acidic Residues Necessary for Pyrophosphate-energized Pumping and Inhibition of the Vacuolar H+-pyrophosphatase byN,N′-Dicyclohexylcarbodiimide

“…However, despite their kinetic equivalence, it is improbable that these results denote a direct interaction between Mg 2ϩ and the residues or domains that undergo covalent modification in all cases. Although the notion of direct protection is consistent with the finding that high affinity Mg 2ϩ protection of the VPPase not only applies to the carboxyl reagents, DCCD and EDAC, but also NEM, a sulfhydryl reagent, and phenylglyoxal, a reagent that preferentially reacts with guanidino side chains (31,32) and the fact that one cytosolic loop, that between transmembrane spans XIII and XIV, containing the Cys residue (Cys 634 ) responsible for substrate-protectable inhibition of the V-PPase by NEM (8,10), also contains Arg, Asp, and Glu residues, it does not reconcile two critical findings. It fails to explain why inhibition by DCCD is second order while EDAC, NEM, and phenylglyoxal inhibit the enzyme with first order kinetics (32) (11).…”

Membrane Na+-pyrophosphatases Can Transport Protons at Low Sodium Concentrations

“…To further characterize the hydrolytic activity of Cl-PPase variants, we analyzed its dependence on the concentrations of the Mg 2 PP i complex, the true substrate of membrane PPase (20,21), at fixed concentrations of Na ϩ (10 mM) and K ϩ (100 mM). In addition to the effects on activity noted above, K m values decreased markedly (7.5-fold) in the S243A variant and less markedly (1.8-fold) in the K681N variant (Table 1).…”

Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

“…4B) (Fig. 2B) (6,7,30). Circular dichroism analysis revealed that K ϩ could elicit the secondary structure variations of H ϩ -PPase (31).…”

“…This unique enzyme hydrolyzes PP i to generate H ϩ -motive force across vacuolar membrane in plants and plasmic membrane in prokaryotes (3,4). In addition, H ϩ -PPase requires Mg 2ϩ ion with PP i as the actual substrate (Mg 2 PP i ) for the hydrolyzing reaction (5,6). Furthermore, K ϩ is essential for stimulating type I H ϩ -PPase; however, Ca 2ϩ , Na ϩ , and F Ϫ inhibit its reaction (7).…”