Abstract
Abstract Vacuolar H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at expense of PPi hydrolysis. The identification of gene encoding an amino acid sequence demonstrates that vacuolar H+-PPase of mung bean contains 6 histidine residues and 15 arginine residues. This study showed vacuolar H+-PPase may contain several histidine and arginine residue(s) essential for the enzymatic activity and H+-translocation of vacuolar H+-PPase. Furthermore, we identified the roles of histidine and arginine residues in mung bean vacuolar H+-PPase by site-directed mutagenesis. A line of mutants with histidine and arginine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among histidine mutants scrutinized, only did the mutation of H716 decrease the enzymatic activity, the proton transport and the coupling ratio of vacuolar H+-PPase. The mutation at H716 of vacuolar H+-PPase shifted the optimum pH value but not the T1/2 (pretreatment temperature at which half enzymatic activity is observed) for PPi hydrolytic activity. Mutation of H716 is obviously declined the effect of substrate protection on the vacuolar H+-PPase as determined by immunoblotting analysis after limited trypsin digestion. Moreover, mutation of these histidine residues modified the inhibitory effects of F- and Na+, but not that of Ca2+. Single substitution of H704, H716 and H758 by alanine released the effect of K+ stimulation, indicating possible location of K+ binding in the vicinity of domains surrounding these residues. A working topological model is thus proposed to elucidate the roles of crucial histidines. As for arginine mutated variants, R242A, R523A, and R609A mutants displayed declined activity of PPi hydrolysis and proton translocation than the wild-type. These mutants showed a shift in optimal pH for enzymatic activity. Among arginine mutants, R242A is relatively resistant to PGO and BD treatments, suggesting that it is the primary target for the attack of these modifiers. Furthermore, only did R242A mutant displayed relatively lower sensitivity to F- inhibition under similar conditions. Taken together, we speculate that R242 may locate in active domain of vacuolar H+-PPase. A working model is proposed to accommodate information from previous studies on the catalytic site of vacuolar H+-PPase.