title: Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase.
abstract: 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.
authors.affiliation: Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, National Tsing Hua University, Hsin Chu 30043, Taiwan.
authors.forename: Chien-Hsien
authors.initials: CH
authors.lastname: Lee
authors.forename: Yih-Jiuan
authors.initials: YJ
authors.lastname: Pan
authors.forename: Yun-Tzu
authors.initials: YT
authors.lastname: Huang
authors.forename: Tseng-Huang
authors.initials: TH
authors.lastname: Liu
authors.forename: Shen-Hsing
authors.initials: SH
authors.lastname: Hsu
authors.forename: Ching-Hung
authors.initials: CH
authors.lastname: Lee
authors.forename: Yen-Wei
authors.initials: YW
authors.lastname: Chen
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Lin
authors.forename: Lin-Kun
authors.initials: LK
authors.lastname: Huang
authors.forename: Rong-Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D043564:Inorganic Pyrophosphatase; D008239:Lysine