Abstract
Vacuolar proton pumping pyrophosphatase (V-PPase; EC 3.6.1.1), a homodimeric proton-translocase sustaining the proton gradient across the membrane, contains a single polypeptide with a molecular mass of approximately 80 kDa. Evidence demonstrated that the C-terminus of V-PPase is relatively conserved in various higher plants and locates near to the catalytic site. However, the real function of C-terminal region is unknown. Our previous study indicated using truncated variants that depletion of last 5 amino acids in C-terminus significantly decreased its activity; however, further removal of next 5 amino acids recovered its activity to about 60-70% of wild type. Accordingly, we identified individual amino acids on C-terminus using site-directed mutagenesis technique that focused on their roles in the structure and function of V-PPase. Our results showed that F755A, G759A, F763A, I765A decreased enzymatic activities, proton translocation, and coupling ratio of V-PPase. Coupling ratios of F755A, G759A retained only about 50% compared to wild type. K+ is stimulatory but Ca2+, F-, and Na+ are inhibitory to the V-PPase. Our results suggested that these C-terminal amino acids are sensitive to various ions especially last 10 residues. For wild type V-PPase, KM and Vmax are about 337 □M and 104 □mol PPi/ mg protein．h, respectively. Mutation of C-termianl amino acids apparently decreased their values of KM and Vmax, indicating a change in their enzymatic reactivity. In conclusion, C-terminal region of plant V-PPase would play an important role on enzymatic mechanism and participate in enzymatic and proton translocation reactions.