Functional and fluorescence analyses of tryptophan residues in H+-pyrophosphatase of Clostridium tetani.

Homodimeric proton-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PPi) hydrolysis and proton translocation. H+-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PPi binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H+-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-Pi binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H+-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H+-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis.

Functional investigation of transmembrane helix 3 in H⁺-translocating pyrophosphatase.

H⁺-translocating pyrophosphatase (H⁺-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PP(i)) hydrolysis. Vigna radiata H⁺-PPase (VrH⁺-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H⁺-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H⁺-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH⁺-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K⁺-mediated stimulation of H⁺-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PP(i) hydrolysis, proton transport, expression, and K⁺ stimulation of H⁺-PPase.

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 (PPi) 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 PPi hydrolytic activity. The effects caused by ions on the activities of WT and 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.