Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

Huang, Yun; Liu, Tseng Huang; Chen, Yen Wei; Lee, Chien Hsien; Chen, Hsueh-Hua; Huang, Tsu Wei; Hsu, Shen-Hsing; Lin, Shih Ming; Pan, Yih Jiuan; Lee, Ching Hung; Hsu, Ian C.; Tseng, Fan Gang; Fu, Chien Chung; Pan, Rong

doi:10.1074/jbc.m110.134916

Cited by 13 publications

(15 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These results were consistent with previous reports that the substrate-binding sites were located at loop 5 between TM5 and TM6 (1,23,26). The binding of IDP tuned the conformational change of H ϩ -PPase to reduce the domain interaction of force peaks 5 and 6, respectively (Fig.…”

Section: Discussionsupporting

confidence: 82%

“…The substrate-induced conformational changes of H ϩ -PPase were described previously (2,7,26), and the precise movements of the active site were further identified by AFM F-Ds. In the presence of substrate PP i , the substrate was bound and hydrolyzed by H ϩ -PPase in the catalytic cycle (2, 17).…”

Section: ϩmentioning

confidence: 99%

“…Enzyme Assay and Protein Determination-PP i hydrolysis activity was determined by measuring the release of P i from PP i as delineated previously (7,12,13,26). The reaction medium contained 30 mM Tris/Mes (pH 8.0), 1 mM MgSO 4 , 0.5 mM NaF, 50 mM KCl, 1 mM PP i , 1.5 g/ml gramicidin D, and 20 -30 g/ml microsomal protein.…”

Section: Cloning Expression and Purification Of His-tagged Hmentioning

confidence: 99%

“…2A). H ϩ -PPase was reconstituted into liposome using the dialysis and Bio-Beads absorption methods modified from previous works (10,26,28,31,32). This reconstitution method aligned all the H ϩ -PPase facing toward the same orientation with comparable specific activity to that on the yeast microsomal membrane (28,33).…”

Section: Characterization Of Purified and Reconstituted Hmentioning

confidence: 99%

“…1 (2,26,30). H ϩ -PPase with His 6 tag at the C terminus was heterologously expressed in yeast and purified into a homogeneous form with high specific activity (7).…”

Section: Characterization Of Purified and Reconstituted Hmentioning

confidence: 99%

See 4 more Smart Citations

Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

Hsu

Liu³

et al. 2015

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Section: Discussionsupporting

confidence: 82%

Section: ϩmentioning

confidence: 99%

Section: Cloning Expression and Purification Of His-tagged Hmentioning

confidence: 99%

Section: Characterization Of Purified and Reconstituted Hmentioning

confidence: 99%

“…1 (2,26,30). H ϩ -PPase with His 6 tag at the C terminus was heterologously expressed in yeast and purified into a homogeneous form with high specific activity (7).…”

Section: Characterization Of Purified and Reconstituted Hmentioning

confidence: 99%

See 3 more Smart Citations

Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

Hsu

Liu³

et al. 2015

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Membrane‐Bound Pyrophosphatases

Kellosalo

Goldman

2015

Encyclopedia of Inorganic and Bioinorganic Chemistry

View full text Add to dashboard Cite

Membrane‐bound pyrophosphatases (M‐PPases) are homodimeric enzymes that couple the vectorial transport of protons and/or sodium ions to pyrophosphate (PP i ) hydrolysis or synthesis. They are found in prokaryotes, plants, and protists and are thus present in all three domains of life. M‐PPases have an important role in conferring tolerance against abiotic stress conditions and are also vital for plant maturation. Like many proteins that catalyze the formation or cleavage of phosphoanhydride bonds (e.g., P‐ and F‐type ATPases, soluble PPases), M‐PPases require magnesium for activity, both because they bind 2–3 free Mg 2+ ions and because the catalytically relevant substrate at physiological pH and Mg ion concentration is Mg 2 PP i . Besides this alkaline earth metal, M‐PPases also bind alkali metals: Na + ‐pumping M‐PPases bind and transport Na + and certain M‐PPases bind and are activated by K + . In this article, we give a concise summary of the current structural and functional understanding of M‐PPases, with an emphasis on describing the role of the metal ions in the enzymatic function of these proteins.

show abstract

Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase

et al. 2013

Self Cite

View full text Add to dashboard Cite

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.Keywords Proton-translocating pyrophosphatase Á Proton transport Á Transmembrane helix Á Site-directed mutagenesis Á Coupling efficiency Á GxxxG-like motif Ching-Hung Lee and Yen-Wei Chen contributed equally to this study.

show abstract

Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

Abstract: Homodimeric

Cited by 13 publications

References 33 publications

Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

Membrane‐Bound Pyrophosphatases

Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase

Contact Info

Product

Resources

About