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Identification of Amino Acid Substitutions that Confer a High Affinity for Sulfaphenazole Binding and a High Catalytic Efficiency for Warfarin Metabolism To P450 2C19
Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant,...
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| Published in: | Biochemistry (Easton) 1998-11, Vol.37 (46), p.16270-16279 |
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| Main Authors: | , , , , , |
| Format: | Article |
| Language: | English |
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| cites | cdi_FETCH-LOGICAL-a348t-4ec6a5781870c312c3fcc2b6409bcceaefa5d2c028c911ff7dcbb26a7c29f9013 |
| container_end_page | 16279 |
| container_issue | 46 |
| container_start_page | 16270 |
| container_title | Biochemistry (Easton) |
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| creator | Jung, Frank Griffin, Keith J Song, Wu Richardson, Toby H Yang, Mierha Johnson, Eric F |
| description | Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, K S, of |
| doi_str_mv | 10.1021/bi981704c |
| format | article |
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Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, K S, of <1 μM. P450 2C19 is not affected by SFZ at concentrations up to 100 μM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227−338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (K S ∼ 4 μM) that was not seen when a shorter fragment of 2C9 was substituted (227−282). However, replacement of amino acids 283−338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a K S < 10 μM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (K S < 5 μM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower K M for (S)-warfarin metabolism (<10 μM) typical of 2C9.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi981704c</identifier><identifier>PMID: 9819219</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amino Acid Sequence ; Amino Acid Substitution - genetics ; Aryl Hydrocarbon Hydroxylases ; Binding, Competitive - genetics ; Catalysis ; Cell Membrane - genetics ; Cell Membrane - metabolism ; Cytochrome P-450 CYP2C19 ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System - genetics ; Cytochrome P-450 Enzyme System - metabolism ; Enzyme Inhibitors - chemistry ; Enzyme Inhibitors - metabolism ; Escherichia coli - genetics ; Genetic Vectors - metabolism ; Humans ; Mixed Function Oxygenases - antagonists & inhibitors ; Mixed Function Oxygenases - genetics ; Mixed Function Oxygenases - metabolism ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - metabolism ; Stereoisomerism ; Sulfaphenazole - metabolism ; Warfarin - chemistry ; Warfarin - metabolism</subject><ispartof>Biochemistry (Easton), 1998-11, Vol.37 (46), p.16270-16279</ispartof><rights>Copyright © 1998 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a348t-4ec6a5781870c312c3fcc2b6409bcceaefa5d2c028c911ff7dcbb26a7c29f9013</citedby><cites>FETCH-LOGICAL-a348t-4ec6a5781870c312c3fcc2b6409bcceaefa5d2c028c911ff7dcbb26a7c29f9013</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9819219$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jung, Frank</creatorcontrib><creatorcontrib>Griffin, Keith J</creatorcontrib><creatorcontrib>Song, Wu</creatorcontrib><creatorcontrib>Richardson, Toby H</creatorcontrib><creatorcontrib>Yang, Mierha</creatorcontrib><creatorcontrib>Johnson, Eric F</creatorcontrib><title>Identification of Amino Acid Substitutions that Confer a High Affinity for Sulfaphenazole Binding and a High Catalytic Efficiency for Warfarin Metabolism To P450 2C19</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, K S, of <1 μM. P450 2C19 is not affected by SFZ at concentrations up to 100 μM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227−338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (K S ∼ 4 μM) that was not seen when a shorter fragment of 2C9 was substituted (227−282). However, replacement of amino acids 283−338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a K S < 10 μM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (K S < 5 μM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower K M for (S)-warfarin metabolism (<10 μM) typical of 2C9.</description><subject>Amino Acid Sequence</subject><subject>Amino Acid Substitution - genetics</subject><subject>Aryl Hydrocarbon Hydroxylases</subject><subject>Binding, Competitive - genetics</subject><subject>Catalysis</subject><subject>Cell Membrane - genetics</subject><subject>Cell Membrane - metabolism</subject><subject>Cytochrome P-450 CYP2C19</subject><subject>Cytochrome P-450 Enzyme Inhibitors</subject><subject>Cytochrome P-450 Enzyme System - genetics</subject><subject>Cytochrome P-450 Enzyme System - metabolism</subject><subject>Enzyme Inhibitors - chemistry</subject><subject>Enzyme Inhibitors - metabolism</subject><subject>Escherichia coli - genetics</subject><subject>Genetic Vectors - metabolism</subject><subject>Humans</subject><subject>Mixed Function Oxygenases - antagonists & inhibitors</subject><subject>Mixed Function Oxygenases - genetics</subject><subject>Mixed Function Oxygenases - metabolism</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Stereoisomerism</subject><subject>Sulfaphenazole - metabolism</subject><subject>Warfarin - chemistry</subject><subject>Warfarin - metabolism</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><recordid>eNptkM9uEzEQhy0EKmnhwAMg-cKhhwXb-9fHsCptRQpFDeK4mp21mykbO7IdifBAPCdbbcmJ02j0fTOj-TH2Ror3Uij5oSfdyFoU-IwtZKlEVmhdPmcLIUSVKV2Jl-w0xoepLURdnLCTSddK6gX7cz0Yl8gSQiLvuLd8uSXn-RJp4Hf7PiZK-0cUedpA4q131gQO_IruN3xpLTlKB259mOzRwm5jHPz2o-EfyQ3k7jm44Z_eQoLxkAj5xTSIZBzOoz8gWAjk-I1J0PuR4pavPb8tSsFVK_Ur9sLCGM3rp3rGvn-6WLdX2err5XW7XGWQF03KCoMVlHUjm1pgLhXmFlH1VSF0j2jAWCgHhUI1qKW0th6w71UFNSpttZD5GTuf92LwMQZju12gLYRDJ0X3GHV3jHpy387ubt9vzXA0n7KdeDZzisn8OmIIP7uqzuuyW9_edZW8-dauPqvuy-S_m33A2D34fXDTp_-5-xfQf5YG</recordid><startdate>19981117</startdate><enddate>19981117</enddate><creator>Jung, Frank</creator><creator>Griffin, Keith J</creator><creator>Song, Wu</creator><creator>Richardson, Toby H</creator><creator>Yang, Mierha</creator><creator>Johnson, Eric F</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></search><sort><creationdate>19981117</creationdate><title>Identification of Amino Acid Substitutions that Confer a High Affinity for Sulfaphenazole Binding and a High Catalytic Efficiency for Warfarin Metabolism To P450 2C19</title><author>Jung, Frank ; Griffin, Keith J ; Song, Wu ; Richardson, Toby H ; Yang, Mierha ; Johnson, Eric F</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a348t-4ec6a5781870c312c3fcc2b6409bcceaefa5d2c028c911ff7dcbb26a7c29f9013</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Amino Acid Sequence</topic><topic>Amino Acid Substitution - genetics</topic><topic>Aryl Hydrocarbon Hydroxylases</topic><topic>Binding, Competitive - genetics</topic><topic>Catalysis</topic><topic>Cell Membrane - genetics</topic><topic>Cell Membrane - metabolism</topic><topic>Cytochrome P-450 CYP2C19</topic><topic>Cytochrome P-450 Enzyme Inhibitors</topic><topic>Cytochrome P-450 Enzyme System - genetics</topic><topic>Cytochrome P-450 Enzyme System - metabolism</topic><topic>Enzyme Inhibitors - chemistry</topic><topic>Enzyme Inhibitors - metabolism</topic><topic>Escherichia coli - genetics</topic><topic>Genetic Vectors - metabolism</topic><topic>Humans</topic><topic>Mixed Function Oxygenases - antagonists & inhibitors</topic><topic>Mixed Function Oxygenases - genetics</topic><topic>Mixed Function Oxygenases - metabolism</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Recombinant Fusion Proteins - biosynthesis</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Stereoisomerism</topic><topic>Sulfaphenazole - metabolism</topic><topic>Warfarin - chemistry</topic><topic>Warfarin - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jung, Frank</creatorcontrib><creatorcontrib>Griffin, Keith J</creatorcontrib><creatorcontrib>Song, Wu</creatorcontrib><creatorcontrib>Richardson, Toby H</creatorcontrib><creatorcontrib>Yang, Mierha</creatorcontrib><creatorcontrib>Johnson, Eric F</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><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jung, Frank</au><au>Griffin, Keith J</au><au>Song, Wu</au><au>Richardson, Toby H</au><au>Yang, Mierha</au><au>Johnson, Eric F</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of Amino Acid Substitutions that Confer a High Affinity for Sulfaphenazole Binding and a High Catalytic Efficiency for Warfarin Metabolism To P450 2C19</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1998-11-17</date><risdate>1998</risdate><volume>37</volume><issue>46</issue><spage>16270</spage><epage>16279</epage><pages>16270-16279</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, K S, of <1 μM. P450 2C19 is not affected by SFZ at concentrations up to 100 μM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227−338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (K S ∼ 4 μM) that was not seen when a shorter fragment of 2C9 was substituted (227−282). However, replacement of amino acids 283−338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a K S < 10 μM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (K S < 5 μM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower K M for (S)-warfarin metabolism (<10 μM) typical of 2C9.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>9819219</pmid><doi>10.1021/bi981704c</doi><tpages>10</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0006-2960 |
| ispartof | Biochemistry (Easton), 1998-11, Vol.37 (46), p.16270-16279 |
| issn | 0006-2960 1520-4995 |
| language | eng |
| recordid | cdi_crossref_primary_10_1021_bi981704c |
| source | American Chemical Society:Jisc Collections:American Chemical Society Read & Publish Agreement 2022-2024 (Reading list) |
| subjects | Amino Acid Sequence Amino Acid Substitution - genetics Aryl Hydrocarbon Hydroxylases Binding, Competitive - genetics Catalysis Cell Membrane - genetics Cell Membrane - metabolism Cytochrome P-450 CYP2C19 Cytochrome P-450 Enzyme Inhibitors Cytochrome P-450 Enzyme System - genetics Cytochrome P-450 Enzyme System - metabolism Enzyme Inhibitors - chemistry Enzyme Inhibitors - metabolism Escherichia coli - genetics Genetic Vectors - metabolism Humans Mixed Function Oxygenases - antagonists & inhibitors Mixed Function Oxygenases - genetics Mixed Function Oxygenases - metabolism Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - metabolism Stereoisomerism Sulfaphenazole - metabolism Warfarin - chemistry Warfarin - metabolism |
| title | Identification of Amino Acid Substitutions that Confer a High Affinity for Sulfaphenazole Binding and a High Catalytic Efficiency for Warfarin Metabolism To P450 2C19 |
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