Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer
Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H+-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The function...
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creator | Huang, Yun-Tzu 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-Long |
description | Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H+-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 ± 4.0 and 67.2 ± 5.7 Å, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding. |
doi_str_mv | 10.1074/jbc.M110.134916 |
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This novel H+-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 ± 4.0 and 67.2 ± 5.7 Å, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M110.134916</identifier><identifier>PMID: 20511234</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Motifs ; Atomic Force Microscopy ; Bioenergetics ; Catalytic Domain ; Clostridium tetani - enzymology ; Dimerization ; Enzymology ; Escherichia coli - enzymology ; Fluorescence Resonance Energy Transfer (FRET) ; Fluorescence Resonance Energy Transfer - methods ; Inorganic Pyrophosphatase - chemistry ; Inorganic Pyrophosphatase - physiology ; Ligands ; Microsomes - metabolism ; Mutation ; Protein Sorting Signals ; Protein Transport ; Proton Pumps ; Proton Transport ; Pyrophosphatases - chemistry ; Site-directed Mutagenesis ; Spectrometry, Fluorescence</subject><ispartof>The Journal of biological chemistry, 2010-07, Vol.285 (31), p.23655-23664</ispartof><rights>2010 © 2010 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>2010 by The American Society for Biochemistry and Molecular Biology, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c532t-a17ac7de83a65e34de775f9523100292a555e251143ac5c14ca36a7b0640631a3</citedby><cites>FETCH-LOGICAL-c532t-a17ac7de83a65e34de775f9523100292a555e251143ac5c14ca36a7b0640631a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911311/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911311/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20511234$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Huang, Yun-Tzu</creatorcontrib><creatorcontrib>Liu, Tseng-Huang</creatorcontrib><creatorcontrib>Chen, Yen-Wei</creatorcontrib><creatorcontrib>Lee, Chien-Hsien</creatorcontrib><creatorcontrib>Chen, Hsueh-Hua</creatorcontrib><creatorcontrib>Huang, Tsu-Wei</creatorcontrib><creatorcontrib>Hsu, Shen-Hsing</creatorcontrib><creatorcontrib>Lin, Shih-Ming</creatorcontrib><creatorcontrib>Pan, Yih-Jiuan</creatorcontrib><creatorcontrib>Lee, Ching-Hung</creatorcontrib><creatorcontrib>Hsu, Ian C.</creatorcontrib><creatorcontrib>Tseng, Fan-Gang</creatorcontrib><creatorcontrib>Fu, Chien-Chung</creatorcontrib><creatorcontrib>Pan, Rong-Long</creatorcontrib><title>Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H+-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 ± 4.0 and 67.2 ± 5.7 Å, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding.</description><subject>Amino Acid Motifs</subject><subject>Atomic Force Microscopy</subject><subject>Bioenergetics</subject><subject>Catalytic Domain</subject><subject>Clostridium tetani - enzymology</subject><subject>Dimerization</subject><subject>Enzymology</subject><subject>Escherichia coli - enzymology</subject><subject>Fluorescence Resonance Energy Transfer (FRET)</subject><subject>Fluorescence Resonance Energy Transfer - methods</subject><subject>Inorganic Pyrophosphatase - chemistry</subject><subject>Inorganic Pyrophosphatase - physiology</subject><subject>Ligands</subject><subject>Microsomes - metabolism</subject><subject>Mutation</subject><subject>Protein Sorting Signals</subject><subject>Protein Transport</subject><subject>Proton Pumps</subject><subject>Proton Transport</subject><subject>Pyrophosphatases - chemistry</subject><subject>Site-directed Mutagenesis</subject><subject>Spectrometry, Fluorescence</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kc1v1DAQxS0EosvCmRv4xgGl9WeyuSBV_UQqAtEWcbMmzmTXVTbe2t5F-9_XIaWCA5Yly_LPb-bNI-QtZ4ecVerorrGHX_h4k6rm5TMy42whC6n5z-dkxpjgRS304oC8ivGO5ZWpl-RAMM25kGpG4qmLCQaL9AcEB8n5IdIG0y_EgR7b5HZIr13CSH1HLz8W3_bBb1Y-blaQICI9xYRh7QZsabOn5_3WB4wWR8HvGP3wW_pswLDc05sAQ-wwvCYvOugjvnk85-T2_Ozm5LK4-nrx-eT4qrBailQAr8BWLS4klBqlarGqdFdrIXk2VgvQWqPIRpQEqy1XFmQJVcNKxUrJQc7Jp0l3s23W2OauUoDebIJbQ9gbD878-zK4lVn6nRE15zLvOfnwKBD8_RZjMmuXzfU9DOi30VRSsTxHrTJ5NJE2-BgDdk9VODNjUiYnZcakzJRU_vHu7-ae-D_RZOD9BHTgDSyDi-b2WjAuGV9UjCudiXoiMA9x5zCYaN04-tYFtMm03v23_AMmyK1H</recordid><startdate>20100730</startdate><enddate>20100730</enddate><creator>Huang, Yun-Tzu</creator><creator>Liu, Tseng-Huang</creator><creator>Chen, Yen-Wei</creator><creator>Lee, Chien-Hsien</creator><creator>Chen, Hsueh-Hua</creator><creator>Huang, Tsu-Wei</creator><creator>Hsu, Shen-Hsing</creator><creator>Lin, Shih-Ming</creator><creator>Pan, Yih-Jiuan</creator><creator>Lee, Ching-Hung</creator><creator>Hsu, Ian C.</creator><creator>Tseng, Fan-Gang</creator><creator>Fu, Chien-Chung</creator><creator>Pan, Rong-Long</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20100730</creationdate><title>Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer</title><author>Huang, Yun-Tzu ; 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-Long</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c532t-a17ac7de83a65e34de775f9523100292a555e251143ac5c14ca36a7b0640631a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Amino Acid Motifs</topic><topic>Atomic Force Microscopy</topic><topic>Bioenergetics</topic><topic>Catalytic Domain</topic><topic>Clostridium tetani - enzymology</topic><topic>Dimerization</topic><topic>Enzymology</topic><topic>Escherichia coli - enzymology</topic><topic>Fluorescence Resonance Energy Transfer (FRET)</topic><topic>Fluorescence Resonance Energy Transfer - methods</topic><topic>Inorganic Pyrophosphatase - chemistry</topic><topic>Inorganic Pyrophosphatase - physiology</topic><topic>Ligands</topic><topic>Microsomes - metabolism</topic><topic>Mutation</topic><topic>Protein Sorting Signals</topic><topic>Protein Transport</topic><topic>Proton Pumps</topic><topic>Proton Transport</topic><topic>Pyrophosphatases - chemistry</topic><topic>Site-directed Mutagenesis</topic><topic>Spectrometry, Fluorescence</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Huang, Yun-Tzu</creatorcontrib><creatorcontrib>Liu, Tseng-Huang</creatorcontrib><creatorcontrib>Chen, Yen-Wei</creatorcontrib><creatorcontrib>Lee, Chien-Hsien</creatorcontrib><creatorcontrib>Chen, Hsueh-Hua</creatorcontrib><creatorcontrib>Huang, Tsu-Wei</creatorcontrib><creatorcontrib>Hsu, Shen-Hsing</creatorcontrib><creatorcontrib>Lin, Shih-Ming</creatorcontrib><creatorcontrib>Pan, Yih-Jiuan</creatorcontrib><creatorcontrib>Lee, Ching-Hung</creatorcontrib><creatorcontrib>Hsu, Ian C.</creatorcontrib><creatorcontrib>Tseng, Fan-Gang</creatorcontrib><creatorcontrib>Fu, Chien-Chung</creatorcontrib><creatorcontrib>Pan, Rong-Long</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Huang, Yun-Tzu</au><au>Liu, Tseng-Huang</au><au>Chen, Yen-Wei</au><au>Lee, Chien-Hsien</au><au>Chen, Hsueh-Hua</au><au>Huang, Tsu-Wei</au><au>Hsu, Shen-Hsing</au><au>Lin, Shih-Ming</au><au>Pan, Yih-Jiuan</au><au>Lee, Ching-Hung</au><au>Hsu, Ian C.</au><au>Tseng, Fan-Gang</au><au>Fu, Chien-Chung</au><au>Pan, Rong-Long</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2010-07-30</date><risdate>2010</risdate><volume>285</volume><issue>31</issue><spage>23655</spage><epage>23664</epage><pages>23655-23664</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H+-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 ± 4.0 and 67.2 ± 5.7 Å, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>20511234</pmid><doi>10.1074/jbc.M110.134916</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Amino Acid Motifs Atomic Force Microscopy Bioenergetics Catalytic Domain Clostridium tetani - enzymology Dimerization Enzymology Escherichia coli - enzymology Fluorescence Resonance Energy Transfer (FRET) Fluorescence Resonance Energy Transfer - methods Inorganic Pyrophosphatase - chemistry Inorganic Pyrophosphatase - physiology Ligands Microsomes - metabolism Mutation Protein Sorting Signals Protein Transport Proton Pumps Proton Transport Pyrophosphatases - chemistry Site-directed Mutagenesis Spectrometry, Fluorescence |
title | Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer |
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