Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.

Abstract Vacuolar H + -translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81 kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae , and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (ΔC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (ΔC10) retained about 60–70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the ΔC10 mutant displayed a shift in T 1/2 (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP i hydrolytic activity. The deletion of the C-terminus substantially modified apparent K + binding constant, but exert no significant changes in the Na + -, F − -, and Ca 2+ -inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K + -regulation of the enzyme in an indirect manner.