Inhibition of plant vacuolar H+-ATPase by diethylpyrocarbonate

Treatment of the tonoplast H+-ATPase from mung bean seedlings (Vigna radiat a L.) with histidine-specific modifier, diethyl pyrocarbonate (DEP), caused a marked loss of the ATP hydrolysis activity and the proton translocation in a concentration-dependent manner. The reaction order of inhibition was c alculated to be 0.98, suggesting that at least one histidine residue of vac uolar H+-ATPase was modified by DEP. The absorbance of the vacuolar H+-ATPa se at 240 nm was progressively increased after incubation with DEP, suggest ing that N-carbethoxyhistidine had been formed. Hydroxylamine, which could break N-carbethoxyhistidine. reversed the absorbance change and partially r estored the enzymic activity. The pK(a) of modified residues of vacuolar H-ATPase was kinetically determined to be 6.73, a value close to that of his tidine. Thus. it is assuredly concluded that histidine residues of the vacu olar H+-ATPase were modified by DEP. Kinetic analysis showed that V-max but not K-m of vacuolar H+-ATPase was decreased by DEP. This result is interpr eted as that the residual activity after DEP inhibition was primarily due t o the unmodified enzyme molecules. Moreover. simultaneous presence of DEP a nd DCCD (N,N ' -dicyclohexyl-carbodiimide), an inhibitor modified at proteo lipid subunit of vacuolar H+-ATPase, did not induce synergistic inhibition, indicating their independent effects. The stoichiometry studies further de monstrate that only one out of four histidine residues modified was involve d in the inhibition of vacuolar H+-ATPase by DEP. Mg2+-ATP, the physiologic al substrate of vacuolar H+-ATPase, but not its analogs, exerted preferenti ally partial protection against DEP, indicating that the histidine residue involved in the inhibition of enzymatic activity may locate at/or near the active site and directly participate in the binding of the substrate. (C) 2 001 Elsevier Science B.V. All rights reserved.