A vacuolar H+-pyrophosphatase (EC 3.6.1.1) that catalyses PPi 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 [14C]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 Asp283, located in the putative loop V of a tentative topological model of vacuolar H+-pyrophosphatase on the cytosolic side, was labelled by radioactive [14C]DCCD. Cytosolic loop V contains both DCCD-sensitive Asp283 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.

This content is only available as a PDF.
You do not currently have access to this content.