Tryptophan Residues at Subunit Interfaces Used as Fluorescence Probes To Investigate Homotropic and Heterotropic Regulation of Aspartate Transcarbamylase
The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications. The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local c...
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Veröffentlicht in: | Biochemistry (Easton) 2001-07, Vol.40 (30), p.8773-8782 |
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description | The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications. The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes. These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation. To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-l-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively. The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation. |
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The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes. These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation. To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-l-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively. The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi0029494</identifier><identifier>PMID: 11467937</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Allosteric Regulation - genetics ; Asparagine - genetics ; Aspartate Carbamoyltransferase - chemistry ; Aspartate Carbamoyltransferase - genetics ; Aspartate Carbamoyltransferase - metabolism ; Aspartic Acid - analogs & derivatives ; Aspartic Acid - metabolism ; Escherichia coli ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Fluorescent Dyes - metabolism ; Kinetics ; Peptide Fragments - chemistry ; Peptide Fragments - genetics ; Peptide Fragments - metabolism ; Phenylalanine - genetics ; Phosphonoacetic Acid - analogs & derivatives ; Phosphonoacetic Acid - metabolism ; Spectrometry, Fluorescence - methods ; Substrate Specificity - genetics ; Titrimetry ; tryptophan ; Tryptophan - chemistry ; Tryptophan - genetics ; Tryptophan - metabolism ; Tyrosine - genetics</subject><ispartof>Biochemistry (Easton), 2001-07, Vol.40 (30), p.8773-8782</ispartof><rights>Copyright © 2001 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a380t-335f345913d7f40857f0d8d791483c8d84abaf3b20b67a0f60effd7e4513cda83</citedby><cites>FETCH-LOGICAL-a380t-335f345913d7f40857f0d8d791483c8d84abaf3b20b67a0f60effd7e4513cda83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi0029494$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi0029494$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11467937$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Fetler, Luc</creatorcontrib><creatorcontrib>Tauc, Patrick</creatorcontrib><creatorcontrib>Hervé, Guy</creatorcontrib><creatorcontrib>Cunin, Raymond</creatorcontrib><creatorcontrib>Brochon, Jean-Claude</creatorcontrib><title>Tryptophan Residues at Subunit Interfaces Used as Fluorescence Probes To Investigate Homotropic and Heterotropic Regulation of Aspartate Transcarbamylase</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications. The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes. These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation. To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-l-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively. The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation.</description><subject>Allosteric Regulation - genetics</subject><subject>Asparagine - genetics</subject><subject>Aspartate Carbamoyltransferase - chemistry</subject><subject>Aspartate Carbamoyltransferase - genetics</subject><subject>Aspartate Carbamoyltransferase - metabolism</subject><subject>Aspartic Acid - analogs & derivatives</subject><subject>Aspartic Acid - metabolism</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Fluorescent Dyes - metabolism</subject><subject>Kinetics</subject><subject>Peptide Fragments - chemistry</subject><subject>Peptide Fragments - genetics</subject><subject>Peptide Fragments - metabolism</subject><subject>Phenylalanine - genetics</subject><subject>Phosphonoacetic Acid - analogs & derivatives</subject><subject>Phosphonoacetic Acid - metabolism</subject><subject>Spectrometry, Fluorescence - methods</subject><subject>Substrate Specificity - genetics</subject><subject>Titrimetry</subject><subject>tryptophan</subject><subject>Tryptophan - chemistry</subject><subject>Tryptophan - genetics</subject><subject>Tryptophan - metabolism</subject><subject>Tyrosine - genetics</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkcFu1DAQhi0EotvCgRdAvoDEITCOnTg5thXtFhVRtak4WpPYLilJnNoO6j4Kb4tXu5QLp9HMfPOP5h9C3jD4yCBnn9oeIK9FLZ6RFStyyERdF8_JCgDKLK9LOCCHIdynVIAUL8kBY6KUNZcr8rvxmzm6-QdO9NqEXi8mUIz0ZmmXqY_0YorGW-xS9TYYTTHQs2Fx3oTOTJ2hV961qde4RP4yIfZ3GA1du9FF7-a-ozhpujZJ5G_h2twtA8beTdRZehxm9HE703icQoe-xXEzYDCvyAuLQzCv9_GI3J59bk7X2eW384vT48sMeQUx47ywXBQ141paAVUhLehKy5qJineVrgS2aHmbQ1tKBFuCsVZLIwrGO40VPyLvd7qzdw_p-qjGPh03DDgZtwTFKgaQXE3ghx3YeReCN1bNvh_RbxQDtf2DevpDYt_uRZd2NPofuTc-AdkO6EM0j0999D9VKbksVHN1o06-5ufrLyff1Xb5ux2PXVD3bvFT8uQ_i_8AS6yg7g</recordid><startdate>20010731</startdate><enddate>20010731</enddate><creator>Fetler, Luc</creator><creator>Tauc, Patrick</creator><creator>Hervé, Guy</creator><creator>Cunin, Raymond</creator><creator>Brochon, Jean-Claude</creator><general>American Chemical Society</general><scope>BSCLL</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>7QL</scope><scope>C1K</scope></search><sort><creationdate>20010731</creationdate><title>Tryptophan Residues at Subunit Interfaces Used as Fluorescence Probes To Investigate Homotropic and Heterotropic Regulation of Aspartate Transcarbamylase</title><author>Fetler, Luc ; Tauc, Patrick ; Hervé, Guy ; Cunin, Raymond ; Brochon, Jean-Claude</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a380t-335f345913d7f40857f0d8d791483c8d84abaf3b20b67a0f60effd7e4513cda83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Allosteric Regulation - genetics</topic><topic>Asparagine - genetics</topic><topic>Aspartate Carbamoyltransferase - chemistry</topic><topic>Aspartate Carbamoyltransferase - genetics</topic><topic>Aspartate Carbamoyltransferase - metabolism</topic><topic>Aspartic Acid - analogs & derivatives</topic><topic>Aspartic Acid - metabolism</topic><topic>Escherichia coli</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Fluorescent Dyes - metabolism</topic><topic>Kinetics</topic><topic>Peptide Fragments - chemistry</topic><topic>Peptide Fragments - genetics</topic><topic>Peptide Fragments - metabolism</topic><topic>Phenylalanine - genetics</topic><topic>Phosphonoacetic Acid - analogs & derivatives</topic><topic>Phosphonoacetic Acid - metabolism</topic><topic>Spectrometry, Fluorescence - methods</topic><topic>Substrate Specificity - genetics</topic><topic>Titrimetry</topic><topic>tryptophan</topic><topic>Tryptophan - chemistry</topic><topic>Tryptophan - genetics</topic><topic>Tryptophan - metabolism</topic><topic>Tyrosine - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Fetler, Luc</creatorcontrib><creatorcontrib>Tauc, Patrick</creatorcontrib><creatorcontrib>Hervé, Guy</creatorcontrib><creatorcontrib>Cunin, Raymond</creatorcontrib><creatorcontrib>Brochon, Jean-Claude</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Fetler, Luc</au><au>Tauc, Patrick</au><au>Hervé, Guy</au><au>Cunin, Raymond</au><au>Brochon, Jean-Claude</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Tryptophan Residues at Subunit Interfaces Used as Fluorescence Probes To Investigate Homotropic and Heterotropic Regulation of Aspartate Transcarbamylase</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2001-07-31</date><risdate>2001</risdate><volume>40</volume><issue>30</issue><spage>8773</spage><epage>8782</epage><pages>8773-8782</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications. The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes. These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation. To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-l-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively. The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>11467937</pmid><doi>10.1021/bi0029494</doi><tpages>10</tpages></addata></record> |
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subjects | Allosteric Regulation - genetics Asparagine - genetics Aspartate Carbamoyltransferase - chemistry Aspartate Carbamoyltransferase - genetics Aspartate Carbamoyltransferase - metabolism Aspartic Acid - analogs & derivatives Aspartic Acid - metabolism Escherichia coli Escherichia coli - enzymology Escherichia coli - genetics Fluorescent Dyes - metabolism Kinetics Peptide Fragments - chemistry Peptide Fragments - genetics Peptide Fragments - metabolism Phenylalanine - genetics Phosphonoacetic Acid - analogs & derivatives Phosphonoacetic Acid - metabolism Spectrometry, Fluorescence - methods Substrate Specificity - genetics Titrimetry tryptophan Tryptophan - chemistry Tryptophan - genetics Tryptophan - metabolism Tyrosine - genetics |
title | Tryptophan Residues at Subunit Interfaces Used as Fluorescence Probes To Investigate Homotropic and Heterotropic Regulation of Aspartate Transcarbamylase |
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