Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis
Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-( o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pat...
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| Veröffentlicht in: | Archives of biochemistry and biophysics 2009-06, Vol.486 (1), p.19-26 |
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| container_title | Archives of biochemistry and biophysics |
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| creator | Czekster, Clarissa M. Neto, Brenno A.D. Lapis, Alexandre A.M. Dupont, Jairton Santos, Diogenes S. Basso, Luiz A. |
| description | Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(
o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for
Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from
M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for
MtIGPS-catalized chemical reaction is proposed. |
| doi_str_mv | 10.1016/j.abb.2009.04.001 |
| format | Article |
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o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for
Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from
M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for
MtIGPS-catalized chemical reaction is proposed.</description><identifier>ISSN: 0003-9861</identifier><identifier>EISSN: 1096-0384</identifier><identifier>DOI: 10.1016/j.abb.2009.04.001</identifier><identifier>PMID: 19364491</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>1-( o-Carboxyphenylamino)-1-deoxyribulose 5-phosphate ; Base Sequence ; Biophysical Phenomena ; Cloning, Molecular ; DNA Primers - genetics ; DNA, Bacterial - genetics ; Drug target ; Genes, Bacterial ; Hydrogen-Ion Concentration ; IGPS ; Indole-3-glycerol phosphate synthase ; Indole-3-Glycerol-Phosphate Synthase - genetics ; Indole-3-Glycerol-Phosphate Synthase - isolation & purification ; Indole-3-Glycerol-Phosphate Synthase - metabolism ; Kinetics ; Magnetic Resonance Spectroscopy ; Models, Chemical ; Mycobacterium tuberculosis ; Mycobacterium tuberculosis - enzymology ; Mycobacterium tuberculosis - genetics ; Mycobacterium tuberculosis - pathogenicity ; Ribulosephosphates - chemical synthesis ; Ribulosephosphates - metabolism ; Spectrometry, Fluorescence ; Spectrometry, Mass, Electrospray Ionization ; Steady-state kinetics ; Thermodynamics ; Tuberculosis ; Virulence</subject><ispartof>Archives of biochemistry and biophysics, 2009-06, Vol.486 (1), p.19-26</ispartof><rights>2009 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c426t-3d6b729e2d43dcb106b03f378027b427cf5b1e3a8487b03432128ec493aa62b83</citedby><cites>FETCH-LOGICAL-c426t-3d6b729e2d43dcb106b03f378027b427cf5b1e3a8487b03432128ec493aa62b83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.abb.2009.04.001$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,777,781,3537,27905,27906,45976</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19364491$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Czekster, Clarissa M.</creatorcontrib><creatorcontrib>Neto, Brenno A.D.</creatorcontrib><creatorcontrib>Lapis, Alexandre A.M.</creatorcontrib><creatorcontrib>Dupont, Jairton</creatorcontrib><creatorcontrib>Santos, Diogenes S.</creatorcontrib><creatorcontrib>Basso, Luiz A.</creatorcontrib><title>Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis</title><title>Archives of biochemistry and biophysics</title><addtitle>Arch Biochem Biophys</addtitle><description>Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(
o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for
Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from
M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for
MtIGPS-catalized chemical reaction is proposed.</description><subject>1-( o-Carboxyphenylamino)-1-deoxyribulose 5-phosphate</subject><subject>Base Sequence</subject><subject>Biophysical Phenomena</subject><subject>Cloning, Molecular</subject><subject>DNA Primers - genetics</subject><subject>DNA, Bacterial - genetics</subject><subject>Drug target</subject><subject>Genes, Bacterial</subject><subject>Hydrogen-Ion Concentration</subject><subject>IGPS</subject><subject>Indole-3-glycerol phosphate synthase</subject><subject>Indole-3-Glycerol-Phosphate Synthase - genetics</subject><subject>Indole-3-Glycerol-Phosphate Synthase - isolation & purification</subject><subject>Indole-3-Glycerol-Phosphate Synthase - metabolism</subject><subject>Kinetics</subject><subject>Magnetic Resonance Spectroscopy</subject><subject>Models, Chemical</subject><subject>Mycobacterium tuberculosis</subject><subject>Mycobacterium tuberculosis - enzymology</subject><subject>Mycobacterium tuberculosis - genetics</subject><subject>Mycobacterium tuberculosis - pathogenicity</subject><subject>Ribulosephosphates - chemical synthesis</subject><subject>Ribulosephosphates - metabolism</subject><subject>Spectrometry, Fluorescence</subject><subject>Spectrometry, Mass, Electrospray Ionization</subject><subject>Steady-state kinetics</subject><subject>Thermodynamics</subject><subject>Tuberculosis</subject><subject>Virulence</subject><issn>0003-9861</issn><issn>1096-0384</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kLtu3DAQRQkjgb1e5wPSGKriSvLwEUpEqsDwC7CRIklpECQ18nIjiRuSCqC_jxa7gDtjilvMubc4hHymUFGg8npbGWsrBqAqEBUAPSErCkqWwBvxgawAgJeqkfSMnKe0XQAqJDslZ1RxKYSiK_LyM6Np5zJlk7H440fM3qUidIUf29BjycvXfnYYQ1_sNiHtNnsuzWPemIRFF8NQPM8uWOMyRj8NRZ4sRjf1Ifl0QT52pk_46Zhr8vvu9tfNQ_n04_7x5vtT6QSTueSttDVTyFrBW2cpSAu843UDrLaC1a77aily04imXj6CM8oadEJxYySzDV-Tq8PuLoa_E6asB58c9r0ZMUxJN0pRIYTck1_eJWXNgdfLrQk9gC6GlCJ2ehf9YOKsKei9fb3Vi329t69B6EXu0rk8jk92wPatcdS9AN8OAC4y_nmMOjmPo8PWR3RZt8G_M_8fxCeVYg</recordid><startdate>20090601</startdate><enddate>20090601</enddate><creator>Czekster, Clarissa M.</creator><creator>Neto, Brenno A.D.</creator><creator>Lapis, Alexandre A.M.</creator><creator>Dupont, Jairton</creator><creator>Santos, Diogenes S.</creator><creator>Basso, Luiz A.</creator><general>Elsevier Inc</general><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>7QL</scope><scope>C1K</scope></search><sort><creationdate>20090601</creationdate><title>Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis</title><author>Czekster, Clarissa M. ; Neto, Brenno A.D. ; Lapis, Alexandre A.M. ; Dupont, Jairton ; Santos, Diogenes S. ; Basso, Luiz A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c426t-3d6b729e2d43dcb106b03f378027b427cf5b1e3a8487b03432128ec493aa62b83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>1-( o-Carboxyphenylamino)-1-deoxyribulose 5-phosphate</topic><topic>Base Sequence</topic><topic>Biophysical Phenomena</topic><topic>Cloning, Molecular</topic><topic>DNA Primers - genetics</topic><topic>DNA, Bacterial - genetics</topic><topic>Drug target</topic><topic>Genes, Bacterial</topic><topic>Hydrogen-Ion Concentration</topic><topic>IGPS</topic><topic>Indole-3-glycerol phosphate synthase</topic><topic>Indole-3-Glycerol-Phosphate Synthase - genetics</topic><topic>Indole-3-Glycerol-Phosphate Synthase - isolation & purification</topic><topic>Indole-3-Glycerol-Phosphate Synthase - metabolism</topic><topic>Kinetics</topic><topic>Magnetic Resonance Spectroscopy</topic><topic>Models, Chemical</topic><topic>Mycobacterium tuberculosis</topic><topic>Mycobacterium tuberculosis - enzymology</topic><topic>Mycobacterium tuberculosis - genetics</topic><topic>Mycobacterium tuberculosis - pathogenicity</topic><topic>Ribulosephosphates - chemical synthesis</topic><topic>Ribulosephosphates - metabolism</topic><topic>Spectrometry, Fluorescence</topic><topic>Spectrometry, Mass, Electrospray Ionization</topic><topic>Steady-state kinetics</topic><topic>Thermodynamics</topic><topic>Tuberculosis</topic><topic>Virulence</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Czekster, Clarissa M.</creatorcontrib><creatorcontrib>Neto, Brenno A.D.</creatorcontrib><creatorcontrib>Lapis, Alexandre A.M.</creatorcontrib><creatorcontrib>Dupont, Jairton</creatorcontrib><creatorcontrib>Santos, Diogenes S.</creatorcontrib><creatorcontrib>Basso, Luiz A.</creatorcontrib><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>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><jtitle>Archives of biochemistry and biophysics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Czekster, Clarissa M.</au><au>Neto, Brenno A.D.</au><au>Lapis, Alexandre A.M.</au><au>Dupont, Jairton</au><au>Santos, Diogenes S.</au><au>Basso, Luiz A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis</atitle><jtitle>Archives of biochemistry and biophysics</jtitle><addtitle>Arch Biochem Biophys</addtitle><date>2009-06-01</date><risdate>2009</risdate><volume>486</volume><issue>1</issue><spage>19</spage><epage>26</epage><pages>19-26</pages><issn>0003-9861</issn><eissn>1096-0384</eissn><abstract>Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(
o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for
Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from
M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for
MtIGPS-catalized chemical reaction is proposed.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>19364491</pmid><doi>10.1016/j.abb.2009.04.001</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | 1-( o-Carboxyphenylamino)-1-deoxyribulose 5-phosphate Base Sequence Biophysical Phenomena Cloning, Molecular DNA Primers - genetics DNA, Bacterial - genetics Drug target Genes, Bacterial Hydrogen-Ion Concentration IGPS Indole-3-glycerol phosphate synthase Indole-3-Glycerol-Phosphate Synthase - genetics Indole-3-Glycerol-Phosphate Synthase - isolation & purification Indole-3-Glycerol-Phosphate Synthase - metabolism Kinetics Magnetic Resonance Spectroscopy Models, Chemical Mycobacterium tuberculosis Mycobacterium tuberculosis - enzymology Mycobacterium tuberculosis - genetics Mycobacterium tuberculosis - pathogenicity Ribulosephosphates - chemical synthesis Ribulosephosphates - metabolism Spectrometry, Fluorescence Spectrometry, Mass, Electrospray Ionization Steady-state kinetics Thermodynamics Tuberculosis Virulence |
| title | Steady-state kinetics of indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis |
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