title: Functional and fluorescence analyses of tryptophan residues in H+-pyrophosphatase of Clostridium tetani.
abstract: 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.
authors.forename: Yen-Wei
authors.initials: YW
authors.lastname: Chen
authors.forename: Ching-Hung
authors.initials: CH
authors.lastname: Lee
authors.forename: Yun-Tzu
authors.initials: YT
authors.lastname: Huang
authors.forename: Yih-Jiuan
authors.initials: YJ
authors.lastname: Pan
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Lin
authors.forename: Yueh-Yu
authors.initials: YY
authors.lastname: Lo
authors.forename: Chien-Hsien
authors.initials: CH
authors.lastname: Lee
authors.forename: Lin-Kun
authors.initials: LK
authors.lastname: Huang
authors.forename: Yu-Fen
authors.initials: YF
authors.lastname: Huang
authors.forename: Yu-Di
authors.initials: YD
authors.lastname: Hsu
authors.forename: Rong-Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D003017:Clostridium tetani / Q000201:enzymology*; D005453:Fluorescence; D043564:Inorganic Pyrophosphatase / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011522:Protons; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D011522:Protons; D014364:Tryptophan; D043564:Inorganic Pyrophosphatase