yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate
The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5'-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the...
Gespeichert in:
Veröffentlicht in: | Journal of bioenergetics and biomembranes 2008-12, Vol.40 (6), p.577-585 |
---|---|
Hauptverfasser: | , , , , , , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
container_end_page | 585 |
---|---|
container_issue | 6 |
container_start_page | 577 |
container_title | Journal of bioenergetics and biomembranes |
container_volume | 40 |
creator | Remani, Sreevidya Sun, Jiakang Kotaria, Rusudan Mayor, June A Brownlee, June M Harrison, David H. T Walters, D. Eric Kaplan, Ronald S |
description | The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5'-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH₂CH₂NH₃ ⁺) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP. |
doi_str_mv | 10.1007/s10863-008-9187-1 |
format | Article |
fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2775541</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>66695316</sourcerecordid><originalsourceid>FETCH-LOGICAL-c522t-ae65f0680906f66031613463220d67833d37663ef0b53ba7024c69e39580f29f3</originalsourceid><addsrcrecordid>eNqFks-u1CAUxhuj8Y5XH8CNEhe6qh6gQHFhYm78l0yiid7EHaEtTLnpQAXG2NfwiaXOxKsudAMk53c-vgNfVd3H8BQDiGcJQ8tpDdDWEreixjeqDWaC1rxt8c1qA7hhdSPk57PqTkpXUEBgcLs6wxKAMME31ffF6JTR3uXQj8EP0ekJ9S5HnQ0qq09ziBnNMWTj_HPkBuOzs67X2QWPgkV5NGi7JOcNiia54WDSepiDT66bDLIhIudH17mfHXszuKI9oG5BH5bohvCt3Mie1PMY0jyW0t3qltVTMvdO-3l1-frVp4u39fb9m3cXL7d1zwjJtTacWeAtSOCWc6CYY9pwSggMXLSUDlRwTo2FjtFOCyBNz6WhkrVgibT0vHpx1J0PXXHVl8GintQc3V7HRQXt1J8V70a1C18VEYKxBheBxyeBGL6UsbPau9SbadLehENSnHPJiq3_ggRTSrlcwUd_gVfhEH15hcIQ3MimXSF8hPoYUorG_rKMQa25UMdcqPLdas2FWq0--H3W645TEApAjkAqJb8z8frmf6k-PDZZHZTeRZfU5UcCmAJmUhIQ9Ae7j88g</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>212149486</pqid></control><display><type>article</type><title>yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate</title><source>MEDLINE</source><source>SpringerLink Journals</source><creator>Remani, Sreevidya ; Sun, Jiakang ; Kotaria, Rusudan ; Mayor, June A ; Brownlee, June M ; Harrison, David H. T ; Walters, D. Eric ; Kaplan, Ronald S</creator><creatorcontrib>Remani, Sreevidya ; Sun, Jiakang ; Kotaria, Rusudan ; Mayor, June A ; Brownlee, June M ; Harrison, David H. T ; Walters, D. Eric ; Kaplan, Ronald S</creatorcontrib><description>The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5'-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH₂CH₂NH₃ ⁺) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP.</description><identifier>ISSN: 0145-479X</identifier><identifier>EISSN: 1573-6881</identifier><identifier>DOI: 10.1007/s10863-008-9187-1</identifier><identifier>PMID: 19002576</identifier><language>eng</language><publisher>Boston: Boston : Springer US</publisher><subject>Animal Anatomy ; Animal Biochemistry ; Binding Sites ; Biochemistry ; Bioorganic Chemistry ; Carrier Proteins - chemistry ; Chemistry ; Chemistry and Materials Science ; Citrate transporter ; Computer Simulation ; Fungal Proteins - metabolism ; Histology ; Inhibition ; Kinetics ; Liposomes ; Lysine - chemistry ; membrane proteins ; Mitochondria ; Mitochondria - metabolism ; Models, Chemical ; Models, Molecular ; Molecular biology ; Morphology ; Organic Chemistry ; Phosphates ; Protein Binding ; Proteins ; Pyridoxal 5'-phosphate ; Pyridoxal Phosphate - chemistry ; Pyridoxal Phosphate - metabolism ; Structure-Activity Relationship ; Substrate binding site ; Substrates ; Translocation ; Yeast ; Yeasts</subject><ispartof>Journal of bioenergetics and biomembranes, 2008-12, Vol.40 (6), p.577-585</ispartof><rights>Springer Science+Business Media, LLC 2008</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c522t-ae65f0680906f66031613463220d67833d37663ef0b53ba7024c69e39580f29f3</citedby><cites>FETCH-LOGICAL-c522t-ae65f0680906f66031613463220d67833d37663ef0b53ba7024c69e39580f29f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s10863-008-9187-1$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s10863-008-9187-1$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,780,784,885,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19002576$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Remani, Sreevidya</creatorcontrib><creatorcontrib>Sun, Jiakang</creatorcontrib><creatorcontrib>Kotaria, Rusudan</creatorcontrib><creatorcontrib>Mayor, June A</creatorcontrib><creatorcontrib>Brownlee, June M</creatorcontrib><creatorcontrib>Harrison, David H. T</creatorcontrib><creatorcontrib>Walters, D. Eric</creatorcontrib><creatorcontrib>Kaplan, Ronald S</creatorcontrib><title>yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate</title><title>Journal of bioenergetics and biomembranes</title><addtitle>J Bioenerg Biomembr</addtitle><addtitle>J Bioenerg Biomembr</addtitle><description>The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5'-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH₂CH₂NH₃ ⁺) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP.</description><subject>Animal Anatomy</subject><subject>Animal Biochemistry</subject><subject>Binding Sites</subject><subject>Biochemistry</subject><subject>Bioorganic Chemistry</subject><subject>Carrier Proteins - chemistry</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Citrate transporter</subject><subject>Computer Simulation</subject><subject>Fungal Proteins - metabolism</subject><subject>Histology</subject><subject>Inhibition</subject><subject>Kinetics</subject><subject>Liposomes</subject><subject>Lysine - chemistry</subject><subject>membrane proteins</subject><subject>Mitochondria</subject><subject>Mitochondria - metabolism</subject><subject>Models, Chemical</subject><subject>Models, Molecular</subject><subject>Molecular biology</subject><subject>Morphology</subject><subject>Organic Chemistry</subject><subject>Phosphates</subject><subject>Protein Binding</subject><subject>Proteins</subject><subject>Pyridoxal 5'-phosphate</subject><subject>Pyridoxal Phosphate - chemistry</subject><subject>Pyridoxal Phosphate - metabolism</subject><subject>Structure-Activity Relationship</subject><subject>Substrate binding site</subject><subject>Substrates</subject><subject>Translocation</subject><subject>Yeast</subject><subject>Yeasts</subject><issn>0145-479X</issn><issn>1573-6881</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNqFks-u1CAUxhuj8Y5XH8CNEhe6qh6gQHFhYm78l0yiid7EHaEtTLnpQAXG2NfwiaXOxKsudAMk53c-vgNfVd3H8BQDiGcJQ8tpDdDWEreixjeqDWaC1rxt8c1qA7hhdSPk57PqTkpXUEBgcLs6wxKAMME31ffF6JTR3uXQj8EP0ekJ9S5HnQ0qq09ziBnNMWTj_HPkBuOzs67X2QWPgkV5NGi7JOcNiia54WDSepiDT66bDLIhIudH17mfHXszuKI9oG5BH5bohvCt3Mie1PMY0jyW0t3qltVTMvdO-3l1-frVp4u39fb9m3cXL7d1zwjJtTacWeAtSOCWc6CYY9pwSggMXLSUDlRwTo2FjtFOCyBNz6WhkrVgibT0vHpx1J0PXXHVl8GintQc3V7HRQXt1J8V70a1C18VEYKxBheBxyeBGL6UsbPau9SbadLehENSnHPJiq3_ggRTSrlcwUd_gVfhEH15hcIQ3MimXSF8hPoYUorG_rKMQa25UMdcqPLdas2FWq0--H3W645TEApAjkAqJb8z8frmf6k-PDZZHZTeRZfU5UcCmAJmUhIQ9Ae7j88g</recordid><startdate>20081201</startdate><enddate>20081201</enddate><creator>Remani, Sreevidya</creator><creator>Sun, Jiakang</creator><creator>Kotaria, Rusudan</creator><creator>Mayor, June A</creator><creator>Brownlee, June M</creator><creator>Harrison, David H. T</creator><creator>Walters, D. Eric</creator><creator>Kaplan, Ronald S</creator><general>Boston : Springer US</general><general>Springer US</general><general>Springer Nature B.V</general><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>3V.</scope><scope>7QO</scope><scope>7QP</scope><scope>7TK</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7P</scope><scope>P64</scope><scope>PDBOC</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>M7N</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20081201</creationdate><title>yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate</title><author>Remani, Sreevidya ; Sun, Jiakang ; Kotaria, Rusudan ; Mayor, June A ; Brownlee, June M ; Harrison, David H. T ; Walters, D. Eric ; Kaplan, Ronald S</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c522t-ae65f0680906f66031613463220d67833d37663ef0b53ba7024c69e39580f29f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Animal Anatomy</topic><topic>Animal Biochemistry</topic><topic>Binding Sites</topic><topic>Biochemistry</topic><topic>Bioorganic Chemistry</topic><topic>Carrier Proteins - chemistry</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Citrate transporter</topic><topic>Computer Simulation</topic><topic>Fungal Proteins - metabolism</topic><topic>Histology</topic><topic>Inhibition</topic><topic>Kinetics</topic><topic>Liposomes</topic><topic>Lysine - chemistry</topic><topic>membrane proteins</topic><topic>Mitochondria</topic><topic>Mitochondria - metabolism</topic><topic>Models, Chemical</topic><topic>Models, Molecular</topic><topic>Molecular biology</topic><topic>Morphology</topic><topic>Organic Chemistry</topic><topic>Phosphates</topic><topic>Protein Binding</topic><topic>Proteins</topic><topic>Pyridoxal 5'-phosphate</topic><topic>Pyridoxal Phosphate - chemistry</topic><topic>Pyridoxal Phosphate - metabolism</topic><topic>Structure-Activity Relationship</topic><topic>Substrate binding site</topic><topic>Substrates</topic><topic>Translocation</topic><topic>Yeast</topic><topic>Yeasts</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Remani, Sreevidya</creatorcontrib><creatorcontrib>Sun, Jiakang</creatorcontrib><creatorcontrib>Kotaria, Rusudan</creatorcontrib><creatorcontrib>Mayor, June A</creatorcontrib><creatorcontrib>Brownlee, June M</creatorcontrib><creatorcontrib>Harrison, David H. T</creatorcontrib><creatorcontrib>Walters, D. Eric</creatorcontrib><creatorcontrib>Kaplan, Ronald S</creatorcontrib><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>ProQuest Central (Corporate)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Materials Science Collection</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of bioenergetics and biomembranes</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Remani, Sreevidya</au><au>Sun, Jiakang</au><au>Kotaria, Rusudan</au><au>Mayor, June A</au><au>Brownlee, June M</au><au>Harrison, David H. T</au><au>Walters, D. Eric</au><au>Kaplan, Ronald S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate</atitle><jtitle>Journal of bioenergetics and biomembranes</jtitle><stitle>J Bioenerg Biomembr</stitle><addtitle>J Bioenerg Biomembr</addtitle><date>2008-12-01</date><risdate>2008</risdate><volume>40</volume><issue>6</issue><spage>577</spage><epage>585</epage><pages>577-585</pages><issn>0145-479X</issn><eissn>1573-6881</eissn><abstract>The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5'-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH₂CH₂NH₃ ⁺) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP.</abstract><cop>Boston</cop><pub>Boston : Springer US</pub><pmid>19002576</pmid><doi>10.1007/s10863-008-9187-1</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
fulltext | fulltext |
identifier | ISSN: 0145-479X |
ispartof | Journal of bioenergetics and biomembranes, 2008-12, Vol.40 (6), p.577-585 |
issn | 0145-479X 1573-6881 |
language | eng |
recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2775541 |
source | MEDLINE; SpringerLink Journals |
subjects | Animal Anatomy Animal Biochemistry Binding Sites Biochemistry Bioorganic Chemistry Carrier Proteins - chemistry Chemistry Chemistry and Materials Science Citrate transporter Computer Simulation Fungal Proteins - metabolism Histology Inhibition Kinetics Liposomes Lysine - chemistry membrane proteins Mitochondria Mitochondria - metabolism Models, Chemical Models, Molecular Molecular biology Morphology Organic Chemistry Phosphates Protein Binding Proteins Pyridoxal 5'-phosphate Pyridoxal Phosphate - chemistry Pyridoxal Phosphate - metabolism Structure-Activity Relationship Substrate binding site Substrates Translocation Yeast Yeasts |
title | yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate |
url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-25T14%3A09%3A59IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=yeast%20mitochondrial%20citrate%20transport%20protein:%20identification%20of%20the%20Lysine%20residues%20responsible%20for%20inhibition%20mediated%20by%20Pyridoxal%205'-phosphate&rft.jtitle=Journal%20of%20bioenergetics%20and%20biomembranes&rft.au=Remani,%20Sreevidya&rft.date=2008-12-01&rft.volume=40&rft.issue=6&rft.spage=577&rft.epage=585&rft.pages=577-585&rft.issn=0145-479X&rft.eissn=1573-6881&rft_id=info:doi/10.1007/s10863-008-9187-1&rft_dat=%3Cproquest_pubme%3E66695316%3C/proquest_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=212149486&rft_id=info:pmid/19002576&rfr_iscdi=true |