Identification of essential lysines involved in substrate binding of vacuolar H⁺-pyrophosphatase

H⁺-translocating pyrophosphatase (H⁺-PPase; EC 3.6.1.1) drives proton transport against an electrochemical potential gradient by hydrolyzing pyrophosphate (PP_i) and is found in various endomembranes of higher plants, bacteria, and some protists. H⁺-PPase contains seven highly conserved lysines. We examined the functional roles of these lysines, which are, for the most part, found in the cytosolic regions of mung bean H⁺-PPase by site-directed mutagenesis. Construction of mutants that each had a cytosolic and highly conserved lysine substituted with an alanine resulted in dramatic drops in the PP_i hydrolytic activity. The effects caused by ions on the activities of WTand mutant H ⁺-PPases suggest that Lys-730 may be in close proximity to the Mg²⁺-binding site, and the great resistance of the K694A and K695A mutants to fluoride inhibition suggests that these lysines are present in the active site. The modifier fluorescein 5′-isothiocyanate (FITC) labeled a lysine at the H⁺-PPase active site but did not inhibit the hydrolytic activities of K250A, K250N, K250T, and K250S, which suggested that Lys-250 is essential for substrate binding and may be involved in proton translocation. Analysis of tryptic digests indicated that Lys-711 and Lys-717 help maintain the conformation of the active site. Proteolytic evidence also demonstrated that Lys-250 is the primary target of trypsin and confirmed its crucial role in H⁺-PPase hydrolysis.