Abstract
The vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) plays a significant role in the maintenance of pH value of cytoplasm via the proton translocation from cytosol to vacuolar lumen at the expense of PPi hydrolysis. The overall structure of the membranes-associated PPases is unclear. According to the topology of V-PPase prediced by TopPred Ⅱ, the motif of catalytic site for substrate hydrolysis was presumably located in loop V and exposed to the cytosol. It was not certain whether the motif of catalytic site interacts with adjacent transmembrane domains (TM), nor is the detailed correlation between the different amino acids. TM VI is relatively conserved and believed to participate in catalytic function and conformation stability. In this study, we carried out alanine scanning mutagenesis along TM VI of the mung bean V-PPase. Different amino acids were substituted singly by alanine and a series of site-direct mutants were constructed, over-expressed in Saccharomyces cerevisiae, and their enzymatic activities and proton translocations were determined. Y299A, E301A, A306S, L307A, L317A and N318A mutants exhibited gross impairment of both PPi hydrolysis and coupling efficiencies. The deterioration of these reactions at both ends and the middle of TM Ⅵ and the location of these amino acids at trisection of the helix wheel suggest their roles in maintaining the stability of conformation. A305S, V308A, A310S, G316A, and H319A exhibited an injury of proton translocation ability and show a decrease in the coupling efficiency but not PPi hydrolysis. They also locate at one face of the helix wheel, implicating their involvement in proton translocation. Studies on ion effects, it showed that the Y299A, C304A, L307A, V308A, L317A and N318A mutants are less sensitive to the K+, Ca2+, Na+, and F-, suggesting they exert effects on binding of these ions. Further evidence indicates that E301 may be involved in binding of these ions but not F-. From the proteolysis analysis, the A305S, A306S, L307A, and N318A mutants were relatively resistant the proteolysis. Moreover, their PPi hydrolysis activities were significantly inhibited expect A305S mutant. We suggest the low enzymatic activity of these mutants is due to the conformational change.