Identification of a Firefly Luciferase Active Site Peptide Using a Benzophenone-based Photooxidation Reagent
Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use...
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Veröffentlicht in: | The Journal of biological chemistry 1997-08, Vol.272 (31), p.19359-19364 |
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container_title | The Journal of biological chemistry |
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creator | Branchini, Bruce R. Magyar, Rachelle A. Marcantonio, Karen M. Newberry, Kate J. Stroh, Justin G. Hinz, Linda K. Murtiashaw, Martha H. |
description | Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen. |
doi_str_mv | 10.1074/jbc.272.31.19359 |
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We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.272.31.19359</identifier><identifier>PMID: 9235934</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Affinity Labels ; Animals ; Binding Sites ; Coleoptera ; Lampyridae ; Luciferases - antagonists & inhibitors ; Luciferases - chemistry ; Luciferases - metabolism ; Oxidation-Reduction ; Photinus pyralis ; Photolysis</subject><ispartof>The Journal of biological chemistry, 1997-08, Vol.272 (31), p.19359-19364</ispartof><rights>1997 © 1997 ASBMB. 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Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.</description><subject>Affinity Labels</subject><subject>Animals</subject><subject>Binding Sites</subject><subject>Coleoptera</subject><subject>Lampyridae</subject><subject>Luciferases - antagonists & inhibitors</subject><subject>Luciferases - chemistry</subject><subject>Luciferases - metabolism</subject><subject>Oxidation-Reduction</subject><subject>Photinus pyralis</subject><subject>Photolysis</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1997</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkTtrHDEUhUVIcNZO-jQBFSHdbKSRxpLcOcYvWLBJYkgnJM3VjszsaD3S-vXrI3uWFAbj26i43zlXnIPQF0rmlAj-49q6eS3qOaNzqlij3qEZJZJVrKF_36MZITWtVN3Ij2g3pWtShiu6g3ZUXWDGZ6g_b2HIwQdncogDjh4bfBJG8P0DXmxc8DCaBPjQ5XAL-HfIgC9hnUML-CqFYVnwnzA8xnUHQxygsoVu8WUXc4z3oZ1cf4FZljOf0Adv-gSft-8eujo5_nN0Vi0uTs-PDheVayjLFXeCN9Y7Ca1xBgSXRnEulBdApOXEUmMtE5550wguKDBJasslSCktJcD20PfJdz3Gmw2krFchOeh7M0DcJC0U3eecqTdBuk-UoOoJJBPoxphSSUevx7Ay44OmRD81oUsTujShGdXPTRTJ1633xq6g_S_YRl_236Z9F5bdXUlc2xBdB6uXNgcTBiWw2wCjTi7A4KAtEpd1G8Prf_gHgJOk3g</recordid><startdate>19970801</startdate><enddate>19970801</enddate><creator>Branchini, Bruce R.</creator><creator>Magyar, Rachelle A.</creator><creator>Marcantonio, Karen M.</creator><creator>Newberry, Kate J.</creator><creator>Stroh, Justin G.</creator><creator>Hinz, Linda K.</creator><creator>Murtiashaw, Martha H.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</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>7SS</scope><scope>7X8</scope></search><sort><creationdate>19970801</creationdate><title>Identification of a Firefly Luciferase Active Site Peptide Using a Benzophenone-based Photooxidation Reagent</title><author>Branchini, Bruce R. ; Magyar, Rachelle A. ; Marcantonio, Karen M. ; Newberry, Kate J. ; Stroh, Justin G. ; Hinz, Linda K. ; Murtiashaw, Martha H.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c513t-4c745bfc8edacae748a94479f7e08b40b1abb37f3fa57471e3802b48e888b10e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1997</creationdate><topic>Affinity Labels</topic><topic>Animals</topic><topic>Binding Sites</topic><topic>Coleoptera</topic><topic>Lampyridae</topic><topic>Luciferases - antagonists & inhibitors</topic><topic>Luciferases - chemistry</topic><topic>Luciferases - metabolism</topic><topic>Oxidation-Reduction</topic><topic>Photinus pyralis</topic><topic>Photolysis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Branchini, Bruce R.</creatorcontrib><creatorcontrib>Magyar, Rachelle A.</creatorcontrib><creatorcontrib>Marcantonio, Karen M.</creatorcontrib><creatorcontrib>Newberry, Kate J.</creatorcontrib><creatorcontrib>Stroh, Justin G.</creatorcontrib><creatorcontrib>Hinz, Linda K.</creatorcontrib><creatorcontrib>Murtiashaw, Martha H.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Entomology Abstracts (Full archive)</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Branchini, Bruce R.</au><au>Magyar, Rachelle A.</au><au>Marcantonio, Karen M.</au><au>Newberry, Kate J.</au><au>Stroh, Justin G.</au><au>Hinz, Linda K.</au><au>Murtiashaw, Martha H.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of a Firefly Luciferase Active Site Peptide Using a Benzophenone-based Photooxidation Reagent</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1997-08-01</date><risdate>1997</risdate><volume>272</volume><issue>31</issue><spage>19359</spage><epage>19364</epage><pages>19359-19364</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. 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subjects | Affinity Labels Animals Binding Sites Coleoptera Lampyridae Luciferases - antagonists & inhibitors Luciferases - chemistry Luciferases - metabolism Oxidation-Reduction Photinus pyralis Photolysis |
title | Identification of a Firefly Luciferase Active Site Peptide Using a Benzophenone-based Photooxidation Reagent |
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