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

Chien Hsien Lee, Yih Jiuan Pan, Yun Tzu Huang, Tseng Huang Liu, Shen Hsing Hsu, Ching Hung Lee, Yen Wei Chen, Shih-Ming Lin, Lin Kun Huang, Rong Long Pan

Research output: Contribution to journalArticle

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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 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.

Original languageEnglish
Pages (from-to)11970-11976
Number of pages7
JournalJournal of Biological Chemistry
Volume286
Issue number14
DOIs
Publication statusPublished - 2011 Apr 8

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Inorganic Pyrophosphatase
Lysine
Substrates
Catalytic Domain
Protons
Mutagenesis
Fluorescein-5-isothiocyanate
Fluorides
Alanine
Trypsin
Site-Directed Mutagenesis
Conformations
Hydrolysis
Bacteria
Binding Sites
Ions

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Lee, C. H., Pan, Y. J., Huang, Y. T., Liu, T. H., Hsu, S. H., Lee, C. H., ... Pan, R. L. (2011). Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase. Journal of Biological Chemistry, 286(14), 11970-11976. https://doi.org/10.1074/jbc.M110.190215
Lee, Chien Hsien ; Pan, Yih Jiuan ; Huang, Yun Tzu ; Liu, Tseng Huang ; Hsu, Shen Hsing ; Lee, Ching Hung ; Chen, Yen Wei ; Lin, Shih-Ming ; Huang, Lin Kun ; Pan, Rong Long. / Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 14. pp. 11970-11976.
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author = "Lee, {Chien Hsien} and Pan, {Yih Jiuan} and Huang, {Yun Tzu} and Liu, {Tseng Huang} and Hsu, {Shen Hsing} and Lee, {Ching Hung} and Chen, {Yen Wei} and Shih-Ming Lin and Huang, {Lin Kun} and Pan, {Rong Long}",
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Lee, CH, Pan, YJ, Huang, YT, Liu, TH, Hsu, SH, Lee, CH, Chen, YW, Lin, S-M, Huang, LK & Pan, RL 2011, 'Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase', Journal of Biological Chemistry, vol. 286, no. 14, pp. 11970-11976. https://doi.org/10.1074/jbc.M110.190215

Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase. / Lee, Chien Hsien; Pan, Yih Jiuan; Huang, Yun Tzu; Liu, Tseng Huang; Hsu, Shen Hsing; Lee, Ching Hung; Chen, Yen Wei; Lin, Shih-Ming; Huang, Lin Kun; Pan, Rong Long.

In: Journal of Biological Chemistry, Vol. 286, No. 14, 08.04.2011, p. 11970-11976.

Research output: Contribution to journalArticle

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T1 - Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase

AU - Lee, Chien Hsien

AU - Pan, Yih Jiuan

AU - Huang, Yun Tzu

AU - Liu, Tseng Huang

AU - Hsu, Shen Hsing

AU - Lee, Ching Hung

AU - Chen, Yen Wei

AU - Lin, Shih-Ming

AU - Huang, Lin Kun

AU - Pan, Rong Long

PY - 2011/4/8

Y1 - 2011/4/8

N2 - 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 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.

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