1-Cys Peroxiredoxin, a Bifunctional Enzyme with Glutathione Peroxidase and Phospholipase A2 Activities
This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional (âmoonlightingâ) enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca 2+ -independent phospholipase A 2 (aiPLA 2 ). The cDNA encoding...
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| Veröffentlicht in: | The Journal of biological chemistry 2000-09, Vol.275 (37), p.28421-28427 |
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| creator | Chen, J W Dodia, C Feinstein, S I Jain, M K Fisher, A B |
| description | This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional (âmoonlightingâ) enzyme with
two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca 2+ -independent phospholipase A 2 (aiPLA 2 ). The cDNA encoding aiPLA 2 âwas found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously
reported E. coli construct which has a His-tag and 50 additional amino acids at the NH 2 terminus, did not exhibit aiPLA 2 activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn -2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H 2 O 2 at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA 2 activity is inhibited by the serine protease inhibitor, diethyl p -nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero- sn -2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx
activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect
on aiPLA 2 activity. Mutation of Ser 32 to Ala abolishes aiPLA 2 activity, yet the NSGPx activity remains unaffected; a Cys 47 to Ser mutant is devoid of peroxidase activity but aiPLA 2 activity remains intact. These results suggest that Ser 32 in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA 2 , while Cys 47 in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of
1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as
well as in protection against oxidative injury. |
| doi_str_mv | 10.1074/jbc.M005073200 |
| format | Article |
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two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca 2+ -independent phospholipase A 2 (aiPLA 2 ). The cDNA encoding aiPLA 2 âwas found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously
reported E. coli construct which has a His-tag and 50 additional amino acids at the NH 2 terminus, did not exhibit aiPLA 2 activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn -2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H 2 O 2 at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA 2 activity is inhibited by the serine protease inhibitor, diethyl p -nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero- sn -2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx
activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect
on aiPLA 2 activity. Mutation of Ser 32 to Ala abolishes aiPLA 2 activity, yet the NSGPx activity remains unaffected; a Cys 47 to Ser mutant is devoid of peroxidase activity but aiPLA 2 activity remains intact. These results suggest that Ser 32 in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA 2 , while Cys 47 in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of
1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as
well as in protection against oxidative injury.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M005073200</identifier><identifier>PMID: 10893423</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Antioxidants - metabolism ; Base Sequence ; Binding Sites ; Glutathione Peroxidase - metabolism ; Humans ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Peroxidases - chemistry ; Peroxidases - metabolism ; Peroxiredoxins ; Phospholipases A - metabolism ; Phospholipases A2 ; Recombinant Proteins - metabolism ; Structure-Activity Relationship</subject><ispartof>The Journal of biological chemistry, 2000-09, Vol.275 (37), p.28421-28427</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c360t-b5f99d404cc02225de459f7a7520d2647eb3c5e469479f77e803b460d107e8e03</citedby><cites>FETCH-LOGICAL-c360t-b5f99d404cc02225de459f7a7520d2647eb3c5e469479f77e803b460d107e8e03</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,777,781,27905,27906</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10893423$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Chen, J W</creatorcontrib><creatorcontrib>Dodia, C</creatorcontrib><creatorcontrib>Feinstein, S I</creatorcontrib><creatorcontrib>Jain, M K</creatorcontrib><creatorcontrib>Fisher, A B</creatorcontrib><title>1-Cys Peroxiredoxin, a Bifunctional Enzyme with Glutathione Peroxidase and Phospholipase A2 Activities</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional (âmoonlightingâ) enzyme with
two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca 2+ -independent phospholipase A 2 (aiPLA 2 ). The cDNA encoding aiPLA 2 âwas found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously
reported E. coli construct which has a His-tag and 50 additional amino acids at the NH 2 terminus, did not exhibit aiPLA 2 activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn -2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H 2 O 2 at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA 2 activity is inhibited by the serine protease inhibitor, diethyl p -nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero- sn -2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx
activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect
on aiPLA 2 activity. Mutation of Ser 32 to Ala abolishes aiPLA 2 activity, yet the NSGPx activity remains unaffected; a Cys 47 to Ser mutant is devoid of peroxidase activity but aiPLA 2 activity remains intact. These results suggest that Ser 32 in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA 2 , while Cys 47 in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of
1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as
well as in protection against oxidative injury.</description><subject>Antioxidants - metabolism</subject><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>Glutathione Peroxidase - metabolism</subject><subject>Humans</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Peroxidases - chemistry</subject><subject>Peroxidases - metabolism</subject><subject>Peroxiredoxins</subject><subject>Phospholipases A - metabolism</subject><subject>Phospholipases A2</subject><subject>Recombinant Proteins - metabolism</subject><subject>Structure-Activity Relationship</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpNkL1PwzAQxS0EoqWwMiIPjKScv-JkLFUpSEV0AInNShyHuMqX4pRS_npcpRLccCe9e-8NP4SuCUwJSH6_SfX0BUCAZBTgBI0JRCxggnycojEAJUFMRTRCF85twA-PyTkaeVPMOGVjlJNgvnd4bbrm23Ym87u-wwl-sPm21r1t6qTEi_pnXxm8s32Bl-W2T_rCP8wxlSXO4KTO8LpoXFs0pW0PyozimS_4sr017hKd5UnpzNXxTtD74-Jt_hSsXpfP89kq0CyEPkhFHscZB641UEpFZriIc5lIQSGjIZcmZVoYHsZcel2aCFjKQ8g8CxMZYBM0HXp11zjXmVy1na2Sbq8IqAMw5YGpP2A-cDME2m1ameyffSDkDbeDobCfxc4jUqltdGEqRaVQTCoacUrYLyeCco0</recordid><startdate>20000915</startdate><enddate>20000915</enddate><creator>Chen, J W</creator><creator>Dodia, C</creator><creator>Feinstein, S I</creator><creator>Jain, M K</creator><creator>Fisher, A B</creator><general>American Society for Biochemistry and Molecular Biology</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></search><sort><creationdate>20000915</creationdate><title>1-Cys Peroxiredoxin, a Bifunctional Enzyme with Glutathione Peroxidase and Phospholipase A2 Activities</title><author>Chen, J W ; Dodia, C ; Feinstein, S I ; Jain, M K ; Fisher, A B</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c360t-b5f99d404cc02225de459f7a7520d2647eb3c5e469479f77e803b460d107e8e03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Antioxidants - metabolism</topic><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>Glutathione Peroxidase - metabolism</topic><topic>Humans</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Peroxidases - chemistry</topic><topic>Peroxidases - metabolism</topic><topic>Peroxiredoxins</topic><topic>Phospholipases A - metabolism</topic><topic>Phospholipases A2</topic><topic>Recombinant Proteins - metabolism</topic><topic>Structure-Activity Relationship</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chen, J W</creatorcontrib><creatorcontrib>Dodia, C</creatorcontrib><creatorcontrib>Feinstein, S I</creatorcontrib><creatorcontrib>Jain, M K</creatorcontrib><creatorcontrib>Fisher, A B</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chen, J W</au><au>Dodia, C</au><au>Feinstein, S I</au><au>Jain, M K</au><au>Fisher, A B</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>1-Cys Peroxiredoxin, a Bifunctional Enzyme with Glutathione Peroxidase and Phospholipase A2 Activities</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2000-09-15</date><risdate>2000</risdate><volume>275</volume><issue>37</issue><spage>28421</spage><epage>28427</epage><pages>28421-28427</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional (âmoonlightingâ) enzyme with
two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca 2+ -independent phospholipase A 2 (aiPLA 2 ). The cDNA encoding aiPLA 2 âwas found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously
reported E. coli construct which has a His-tag and 50 additional amino acids at the NH 2 terminus, did not exhibit aiPLA 2 activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn -2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H 2 O 2 at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA 2 activity is inhibited by the serine protease inhibitor, diethyl p -nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero- sn -2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx
activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect
on aiPLA 2 activity. Mutation of Ser 32 to Ala abolishes aiPLA 2 activity, yet the NSGPx activity remains unaffected; a Cys 47 to Ser mutant is devoid of peroxidase activity but aiPLA 2 activity remains intact. These results suggest that Ser 32 in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA 2 , while Cys 47 in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of
1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as
well as in protection against oxidative injury.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>10893423</pmid><doi>10.1074/jbc.M005073200</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Antioxidants - metabolism Base Sequence Binding Sites Glutathione Peroxidase - metabolism Humans Molecular Sequence Data Mutagenesis, Site-Directed Peroxidases - chemistry Peroxidases - metabolism Peroxiredoxins Phospholipases A - metabolism Phospholipases A2 Recombinant Proteins - metabolism Structure-Activity Relationship |
| title | 1-Cys Peroxiredoxin, a Bifunctional Enzyme with Glutathione Peroxidase and Phospholipase A2 Activities |
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