Localization of a carboxylic residue possibly involved in the inhibition of vacuolar H+-pyrophosphatase by N,N′-dicyclohexylcarbodi-imide

Abstract

A vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) that catalyses PP(i) hydrolysis and the electrogenic translocation of protons from the cytosol to the vacuole lumen, was purified from etiolated hypocotyls of mung bean seedlings (Vigna radiata L.). Group-specific modification was used to identify a carboxylic residue involved in the inhibition of vacuolar H(+)-pyrophosphatase. Carbodi-imides, such as N,N'-dicyclohexylcarbodi-imide (DCCD) and 1-ethyl-3-(3-dimethylamino-propyl)carbodi-imide, and Woodward's reagent K caused a progressive decline in the enzymic activity of vacuolar H(+)-pyrophosphatase in a time- and concentration-dependent manner. The stoichiometry of labelling of the vacuolar H(+)-pyrophosphatase by [(14)C]DCCD determined that DCCD modifies one carboxylic residue per subunit of the enzyme. Protection studies suggest that the DCCD-reactive carboxylic residue resides at or near the substrate-binding site. Furthermore, peptide mapping analysis reveals that Asp(283), located in the putative loop V of a tentative topological model of vacuolar H(+)-pyrophosphatase on the cytosolic side, was labelled by radioactive [(14)C]DCCD. Cytosolic loop V contains both DCCD-sensitive Asp(283) and a conserved motif sequence, rendering it a candidate for the catalytic site of vacuolar H(+)-pyrophosphatase. A topological picture of the active domain of vacuolar H(+)-pyrophosphatase is tentatively proposed.

Inorganic pyrophosphatases: One substrate, three mechanisms

“…The active site is closed by a long loop between TM 5 and 6 (residues 206–225 in TmPPase) on substrate binding, and opened again after the hydrolysis and pumping have occurred (see below) [12,19]. This loop movement explains the results of FRET measurements and the protection of conserved residues from modification upon ligand binding [36–42] (see above).…”

“…Mutational analysis and sequence comparison identified three conserved cytoplasmic “acidic motifs” necessary for activity between TM helices 5 and 6, 11 and 12, and 15 and 16. FRET‐measurements [36], and studies with covalent inhibitors [37–42] showed that ligand binding causes the movement of the conserved motifs and the protection of various conserved residues (TmPPase: R191, K199, D232, K499, C599 and H681).…”

Radiation inactivation analysis of H+‐pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings

“…Preparation of tonoplast was carried out according to Maeshima and Yoshida with minor modifications as described previously [20,21].…”

Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase

“…1b). Functional analyses authentically indicated that several residues in the inner wall are crucial for PP i hydrolysis and proton translocation (Yang et al 1999;Nakanishi et al 2001; Van et al 2005;Pan et al 2011;Lin et al 2012); however, detailed information on the structure/function relationship of the outer wall has yet to be validated.…”