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

A vacuolar H+-pyrophosphatase (EC 3.6.1.1) that catalyses PPi hydrolysis an d the electrogenic translocation of protons from the cytosol to the vacuole lumen, was purified from etiolated hypocotyls of mung bean seedlings (Vign a radiata L.). Group-specific modification was used to identify a carboxyli c residue involved in the inhibition of vacuolar H+-pyrophosphatase. Carbod i-imides, such as N,N'-dicyclohexylcarbodi-imide (DCCD) and 1-ethyl-3-(3-di methylamino-propyl)carbo and Woodward's reagent K caused a progressive decl ine in the enzymic activity of vacuolar H+-pyrophosphatase in a time- and c oncentration-dependent manner. The stoichiometry of labelling of the vacuol ar H+-pyrophosphatase by [C-14]DCCD determined that DCCD modifies one carbo xylic residue per subunit of the enzyme. Protection studies suggest that th e 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+-pyro phosphatase on the cytosolic side, was labelled by radioactive [C-14]DCCD. Cytosolic loop V contains both DCCD-sensitive Asp(283) and a conserved moti f 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.