Ability of Tetrahydrobiopterin Analogues to Support Catalysis by Inducible Nitric Oxide Synthase: Formation of a Pterin Radical Is Required for Enzyme Activity
Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H4B) or tetrahydrobiopterin analogues (5-methyl-H4B and 4-amino-H4B), and the ability of bound 5-methyl-H4B and 4-amino-H4B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme dom...
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| Veröffentlicht in: | Biochemistry (Easton) 2003-11, Vol.42 (45), p.13287-13303 |
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| creator | Hurshman, Amy R Krebs, Carsten Edmondson, Dale E Marletta, Michael A |
| description | Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H4B) or tetrahydrobiopterin analogues (5-methyl-H4B and 4-amino-H4B), and the ability of bound 5-methyl-H4B and 4-amino-H4B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiNOS) was examined. In a single turnover with HDiNOS, 5-methyl-H4B forms a very stable radical, 5-methyl-H3B•, that accumulates in the arginine reaction to ∼60% of the HDiNOS concentration and decays ∼400-fold more slowly than H3B• (0.0003 vs 0.12 s-1). The amount of radical (5-methyl-H3B• or H3B•) observed in the NHA reaction is very small ( |
| doi_str_mv | 10.1021/bi035491p |
| format | Article |
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In a single turnover with HDiNOS, 5-methyl-H4B forms a very stable radical, 5-methyl-H3B•, that accumulates in the arginine reaction to ∼60% of the HDiNOS concentration and decays ∼400-fold more slowly than H3B• (0.0003 vs 0.12 s-1). The amount of radical (5-methyl-H3B• or H3B•) observed in the NHA reaction is very small (<3% of HDiNOS). The activity of 5-methyl-H4B-saturated FLiNOS and HDiNOS is similar to that when H4B is bound: arginine is hydroxylated to NHA, and NHA is oxidized exclusively to citrulline and •NO. A pterin radical was not observed with 4-amino-H4B- or pterin-free HDiNOS with either substrate. The catalytic activity of 4-amino-H4B-bound FLiNOS and HDiNOS resembles that of pterin-free iNOS: the hydroxylation of arginine is very unfavorable (<2% that of H4B-bound iNOS), and NHA is oxidized to a mixture of amino acid products (citrulline and cyanoornithine) and NO- rather than •NO. These results demonstrate that the bound pterin cofactor undergoes a one-electron oxidation (to form a pterin radical), which is essential to its ability to support normal NOS turnover. Although binding of H4B also stabilizes the NOS structure and active site, the most critical role of the pterin cofactor in NOS appears to be in electron transfer.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi035491p</identifier><identifier>PMID: 14609340</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amino Acids - analysis ; Arginine - analogs & derivatives ; Arginine - chemistry ; Binding Sites ; Biopterins - analogs & derivatives ; Biopterins - chemistry ; Catalysis ; Dithionite - chemistry ; Electron Spin Resonance Spectroscopy ; Enzyme Activation ; Free Radicals - chemistry ; Freezing ; Heme - chemistry ; Nitrates - analysis ; Nitric Oxide Synthase - chemistry ; Nitric Oxide Synthase Type II ; Nitrites - analysis ; Oxyhemoglobins - chemistry ; Protein Structure, Tertiary ; Pterins - chemistry ; Recombinant Proteins - chemistry ; Time Factors</subject><ispartof>Biochemistry (Easton), 2003-11, Vol.42 (45), p.13287-13303</ispartof><rights>Copyright © 2003 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a349t-d1e2516e035285e940bffcc4d62db6c4a9fdca0ce2373088f8ed26e47220ded43</citedby><cites>FETCH-LOGICAL-a349t-d1e2516e035285e940bffcc4d62db6c4a9fdca0ce2373088f8ed26e47220ded43</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/bi035491p$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi035491p$$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/14609340$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hurshman, Amy R</creatorcontrib><creatorcontrib>Krebs, Carsten</creatorcontrib><creatorcontrib>Edmondson, Dale E</creatorcontrib><creatorcontrib>Marletta, Michael A</creatorcontrib><title>Ability of Tetrahydrobiopterin Analogues to Support Catalysis by Inducible Nitric Oxide Synthase: Formation of a Pterin Radical Is Required for Enzyme Activity</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H4B) or tetrahydrobiopterin analogues (5-methyl-H4B and 4-amino-H4B), and the ability of bound 5-methyl-H4B and 4-amino-H4B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiNOS) was examined. In a single turnover with HDiNOS, 5-methyl-H4B forms a very stable radical, 5-methyl-H3B•, that accumulates in the arginine reaction to ∼60% of the HDiNOS concentration and decays ∼400-fold more slowly than H3B• (0.0003 vs 0.12 s-1). The amount of radical (5-methyl-H3B• or H3B•) observed in the NHA reaction is very small (<3% of HDiNOS). The activity of 5-methyl-H4B-saturated FLiNOS and HDiNOS is similar to that when H4B is bound: arginine is hydroxylated to NHA, and NHA is oxidized exclusively to citrulline and •NO. A pterin radical was not observed with 4-amino-H4B- or pterin-free HDiNOS with either substrate. The catalytic activity of 4-amino-H4B-bound FLiNOS and HDiNOS resembles that of pterin-free iNOS: the hydroxylation of arginine is very unfavorable (<2% that of H4B-bound iNOS), and NHA is oxidized to a mixture of amino acid products (citrulline and cyanoornithine) and NO- rather than •NO. These results demonstrate that the bound pterin cofactor undergoes a one-electron oxidation (to form a pterin radical), which is essential to its ability to support normal NOS turnover. Although binding of H4B also stabilizes the NOS structure and active site, the most critical role of the pterin cofactor in NOS appears to be in electron transfer.</description><subject>Amino Acids - analysis</subject><subject>Arginine - analogs & derivatives</subject><subject>Arginine - chemistry</subject><subject>Binding Sites</subject><subject>Biopterins - analogs & derivatives</subject><subject>Biopterins - chemistry</subject><subject>Catalysis</subject><subject>Dithionite - chemistry</subject><subject>Electron Spin Resonance Spectroscopy</subject><subject>Enzyme Activation</subject><subject>Free Radicals - chemistry</subject><subject>Freezing</subject><subject>Heme - chemistry</subject><subject>Nitrates - analysis</subject><subject>Nitric Oxide Synthase - chemistry</subject><subject>Nitric Oxide Synthase Type II</subject><subject>Nitrites - analysis</subject><subject>Oxyhemoglobins - chemistry</subject><subject>Protein Structure, Tertiary</subject><subject>Pterins - chemistry</subject><subject>Recombinant Proteins - chemistry</subject><subject>Time Factors</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkcFu1DAQhi0EokvhwAsgX0DiELAdx9lw2666sFJFq83Sq-XYE-qSxKntoKanXnkNHo0nIVVW5cJpNJpP_z8zP0KvKflACaMfK0vSjBe0f4IWNGMk4UWRPUULQohIWCHIEXoRwvXUcpLz5-iIckGKlJMF-r2qbGPjiF2N9xC9uhqNd5V1fQRvO7zqVOO-DxBwdLgc-t75iNcqqmYMNuBqxNvODNpWDeCvNnqr8fmtNYDLsYtXKsCnP_e_8Mb5VkXrugcbhS9m7Z0yVqsGbwPewc1gPRhcO49Pu7uxBbzS0f6cVnuJntWqCfDqUI_Rt83pfv0lOTv_vF2vzhKV8iImhgLLqIDpFWyZQcFJVddacyOYqYTmqqiNVkQDS_OULJf1EgwTwHPGiAHD02P0btbtvbuZLo6ytUFD06gO3BBkTlPBBGcT-H4GtXcheKhl722r_CgpkQ-ByMdAJvbNQXSoWjD_yEMCE5DMgA0Rbh_nyv-QIk_zTO4vSin4ZXlSXm7kbuLfzrzSQV67wU8Bhf8Y_wWshqVB</recordid><startdate>20031118</startdate><enddate>20031118</enddate><creator>Hurshman, Amy R</creator><creator>Krebs, Carsten</creator><creator>Edmondson, Dale E</creator><creator>Marletta, Michael A</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>7X8</scope></search><sort><creationdate>20031118</creationdate><title>Ability of Tetrahydrobiopterin Analogues to Support Catalysis by Inducible Nitric Oxide Synthase: Formation of a Pterin Radical Is Required for Enzyme Activity</title><author>Hurshman, Amy R ; Krebs, Carsten ; Edmondson, Dale E ; Marletta, Michael A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a349t-d1e2516e035285e940bffcc4d62db6c4a9fdca0ce2373088f8ed26e47220ded43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Amino Acids - analysis</topic><topic>Arginine - analogs & derivatives</topic><topic>Arginine - chemistry</topic><topic>Binding Sites</topic><topic>Biopterins - analogs & derivatives</topic><topic>Biopterins - chemistry</topic><topic>Catalysis</topic><topic>Dithionite - chemistry</topic><topic>Electron Spin Resonance Spectroscopy</topic><topic>Enzyme Activation</topic><topic>Free Radicals - chemistry</topic><topic>Freezing</topic><topic>Heme - chemistry</topic><topic>Nitrates - analysis</topic><topic>Nitric Oxide Synthase - chemistry</topic><topic>Nitric Oxide Synthase Type II</topic><topic>Nitrites - analysis</topic><topic>Oxyhemoglobins - chemistry</topic><topic>Protein Structure, Tertiary</topic><topic>Pterins - chemistry</topic><topic>Recombinant Proteins - chemistry</topic><topic>Time Factors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hurshman, Amy R</creatorcontrib><creatorcontrib>Krebs, Carsten</creatorcontrib><creatorcontrib>Edmondson, Dale E</creatorcontrib><creatorcontrib>Marletta, Michael A</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>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hurshman, Amy R</au><au>Krebs, Carsten</au><au>Edmondson, Dale E</au><au>Marletta, Michael A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Ability of Tetrahydrobiopterin Analogues to Support Catalysis by Inducible Nitric Oxide Synthase: Formation of a Pterin Radical Is Required for Enzyme Activity</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2003-11-18</date><risdate>2003</risdate><volume>42</volume><issue>45</issue><spage>13287</spage><epage>13303</epage><pages>13287-13303</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Pterin-free inducible nitric oxide synthase (iNOS) was reconstituted with tetrahydrobiopterin (H4B) or tetrahydrobiopterin analogues (5-methyl-H4B and 4-amino-H4B), and the ability of bound 5-methyl-H4B and 4-amino-H4B to support catalysis by either full-length iNOS (FLiNOS) or the isolated heme domain (HDiNOS) was examined. In a single turnover with HDiNOS, 5-methyl-H4B forms a very stable radical, 5-methyl-H3B•, that accumulates in the arginine reaction to ∼60% of the HDiNOS concentration and decays ∼400-fold more slowly than H3B• (0.0003 vs 0.12 s-1). The amount of radical (5-methyl-H3B• or H3B•) observed in the NHA reaction is very small (<3% of HDiNOS). The activity of 5-methyl-H4B-saturated FLiNOS and HDiNOS is similar to that when H4B is bound: arginine is hydroxylated to NHA, and NHA is oxidized exclusively to citrulline and •NO. A pterin radical was not observed with 4-amino-H4B- or pterin-free HDiNOS with either substrate. The catalytic activity of 4-amino-H4B-bound FLiNOS and HDiNOS resembles that of pterin-free iNOS: the hydroxylation of arginine is very unfavorable (<2% that of H4B-bound iNOS), and NHA is oxidized to a mixture of amino acid products (citrulline and cyanoornithine) and NO- rather than •NO. These results demonstrate that the bound pterin cofactor undergoes a one-electron oxidation (to form a pterin radical), which is essential to its ability to support normal NOS turnover. Although binding of H4B also stabilizes the NOS structure and active site, the most critical role of the pterin cofactor in NOS appears to be in electron transfer.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>14609340</pmid><doi>10.1021/bi035491p</doi><tpages>17</tpages></addata></record> |
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| ispartof | Biochemistry (Easton), 2003-11, Vol.42 (45), p.13287-13303 |
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| source | MEDLINE; ACS Publications |
| subjects | Amino Acids - analysis Arginine - analogs & derivatives Arginine - chemistry Binding Sites Biopterins - analogs & derivatives Biopterins - chemistry Catalysis Dithionite - chemistry Electron Spin Resonance Spectroscopy Enzyme Activation Free Radicals - chemistry Freezing Heme - chemistry Nitrates - analysis Nitric Oxide Synthase - chemistry Nitric Oxide Synthase Type II Nitrites - analysis Oxyhemoglobins - chemistry Protein Structure, Tertiary Pterins - chemistry Recombinant Proteins - chemistry Time Factors |
| title | Ability of Tetrahydrobiopterin Analogues to Support Catalysis by Inducible Nitric Oxide Synthase: Formation of a Pterin Radical Is Required for Enzyme Activity |
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