A Continuous, Anaerobic Spectrophotometric Assay for Chorismate Synthase Activity That Utilizes Photoreduced Flavin Mononucleotide
A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (FMNH 2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 n...
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| Veröffentlicht in: | Analytical biochemistry 1994-07, Vol.220 (1), p.137-141 |
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| container_title | Analytical biochemistry |
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| creator | Ramjee, M.K. Coggins, J.R. Thorneley, R.N.F. |
| description | A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (FMNH
2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of ±2 μM product. The assay conditions have been optimized with respect to FMNH
2 (cofactor), EPSP (substrate) and enzyme concentrations, buffer type, and pH. The potential of the assay for detailed steady-state kinetic studies to elucidate the mechanism of action of this commercially important enzyme is also demonstrated. |
| doi_str_mv | 10.1006/abio.1994.1309 |
| format | Article |
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2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of ±2 μM product. The assay conditions have been optimized with respect to FMNH
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2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of ±2 μM product. The assay conditions have been optimized with respect to FMNH
2 (cofactor), EPSP (substrate) and enzyme concentrations, buffer type, and pH. The potential of the assay for detailed steady-state kinetic studies to elucidate the mechanism of action of this commercially important enzyme is also demonstrated.</description><subject>Anaerobiosis</subject><subject>Escherichia coli - enzymology</subject><subject>Flavin Mononucleotide - chemistry</subject><subject>Kinetics</subject><subject>Lyases - analysis</subject><subject>Oxidation-Reduction</subject><subject>Phosphorus-Oxygen Lyases</subject><subject>Reproducibility of Results</subject><subject>Spectrophotometry, Ultraviolet</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1994</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kEFr3DAQhUVJSbZpr70FdMqp3kqWI1lHsyRtIKWFJGchS2NWxZY2krywPeaXV2aX3HKaYd6bB-9D6Csla0oI_657F9ZUymZNGZEf0IoSyStS9jO0IoSwquZSXKBPKf0lhNLmhp-jcyFFWzO-Qq8d3gSfnZ_DnL7hzmuIoXcGP-7A5Bh225DDBDmWU5eSPuAhRLzZhujSpDPgx4PPW50Adya7vcsH_LTVGT9nN7p_kPCfJSCCnQ1YfDfqvfP4V_DBz2aEkJ2Fz-jjoMcEX07zEj3f3T5tflYPv3_cb7qHyjAmc9WXtg1rh4FoqMEQzm0_CNoLqiW3mlthh5o2ZfSMNrrRdClua0FEL60m7BJdH3N3MbzMkLKaXDIwjtpDKa8Eb28Ea1kxro9GE0NKEQa1i27S8aAoUQt0tUBXC3S1QC8PV6fkuZ_AvtlPlIveHnUo9fYOokrGgS9EXCyUlQ3uvej_ZaKUCA</recordid><startdate>19940701</startdate><enddate>19940701</enddate><creator>Ramjee, M.K.</creator><creator>Coggins, J.R.</creator><creator>Thorneley, R.N.F.</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></search><sort><creationdate>19940701</creationdate><title>A Continuous, Anaerobic Spectrophotometric Assay for Chorismate Synthase Activity That Utilizes Photoreduced Flavin Mononucleotide</title><author>Ramjee, M.K. ; Coggins, J.R. ; Thorneley, R.N.F.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c339t-b006438ff0ae2ec066dbf71b71a96da6d7df214d7db314a4a12697d2707b9da03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1994</creationdate><topic>Anaerobiosis</topic><topic>Escherichia coli - enzymology</topic><topic>Flavin Mononucleotide - chemistry</topic><topic>Kinetics</topic><topic>Lyases - analysis</topic><topic>Oxidation-Reduction</topic><topic>Phosphorus-Oxygen Lyases</topic><topic>Reproducibility of Results</topic><topic>Spectrophotometry, Ultraviolet</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ramjee, M.K.</creatorcontrib><creatorcontrib>Coggins, J.R.</creatorcontrib><creatorcontrib>Thorneley, R.N.F.</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><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ramjee, M.K.</au><au>Coggins, J.R.</au><au>Thorneley, R.N.F.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A Continuous, Anaerobic Spectrophotometric Assay for Chorismate Synthase Activity That Utilizes Photoreduced Flavin Mononucleotide</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1994-07-01</date><risdate>1994</risdate><volume>220</volume><issue>1</issue><spage>137</spage><epage>141</epage><pages>137-141</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (FMNH
2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of ±2 μM product. The assay conditions have been optimized with respect to FMNH
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| ispartof | Analytical biochemistry, 1994-07, Vol.220 (1), p.137-141 |
| issn | 0003-2697 1096-0309 |
| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals Complete |
| subjects | Anaerobiosis Escherichia coli - enzymology Flavin Mononucleotide - chemistry Kinetics Lyases - analysis Oxidation-Reduction Phosphorus-Oxygen Lyases Reproducibility of Results Spectrophotometry, Ultraviolet |
| title | A Continuous, Anaerobic Spectrophotometric Assay for Chorismate Synthase Activity That Utilizes Photoreduced Flavin Mononucleotide |
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