Catalysis of Oxidative Protein Folding by Mutants of Protein Disulfide Isomerase with a Single Active-Site Cysteine
Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol...
Gespeichert in:
| Veröffentlicht in: | Biochemistry (Easton) 1996-02, Vol.35 (6), p.1972-1980 |
|---|---|
| Hauptverfasser: | , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 1980 |
|---|---|
| container_issue | 6 |
| container_start_page | 1972 |
| container_title | Biochemistry (Easton) |
| container_volume | 35 |
| creator | Walker, Kenneth W Lyles, Michelle M Gilbert, Hiram F |
| description | Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946−30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 μM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%−50% of the k cat activity of wild-type active sites during the rearrangement phase of RNase refolding but |
| doi_str_mv | 10.1021/bi952157n |
| format | Article |
| fullrecord | <record><control><sourceid>istex_cross</sourceid><recordid>TN_cdi_crossref_primary_10_1021_bi952157n</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>ark_67375_TPS_ZW0FWHGR_W</sourcerecordid><originalsourceid>FETCH-LOGICAL-a348t-86c1e79ab9b12720ad537a2718727cca149464dc2d6dcc37eda3d28fb04e17a53</originalsourceid><addsrcrecordid>eNptkEtPwzAQhC0EKqVw4Acg-cKBQ8B2Hk6OVaEPqagVLarExdrYDrikSRUn0Px7UlJ64rRazTe7mkHompJ7Shh9iE3kM-rz7AR1qc-I40WRf4q6hJDAYVFAztGFtetm9Qj3OqgTBm4UhLSL7ABKSGtrLM4TPNsZBaX50nhe5KU2GR7mqTLZO45r_FyVkJW_3J_6aGyVJkZpPLH5RhdgNf425QcGvGhcqcZ9uT_nLEyp8aC2e5e-RGcJpFZfHWYPvQ6floOxM52NJoP-1AHXC0snDCTVPII4iinjjIDyXQ6M05AzLiVQL_ICT0mmAiWly7UCV7EwiYmnKQff7aG79q4scmsLnYhtYTZQ1IISse9NHHtr2JuW3VbxRqsjeSiq0Z1WN02G3VGG4lME3OW-WM4X4m1Fhqvx6EWsGv625UFasc6rImuS_vP3B_YKhOg</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype></control><display><type>article</type><title>Catalysis of Oxidative Protein Folding by Mutants of Protein Disulfide Isomerase with a Single Active-Site Cysteine</title><source>MEDLINE</source><source>American Chemical Society Journals</source><creator>Walker, Kenneth W ; Lyles, Michelle M ; Gilbert, Hiram F</creator><creatorcontrib>Walker, Kenneth W ; Lyles, Michelle M ; Gilbert, Hiram F</creatorcontrib><description>Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946−30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 μM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%−50% of the k cat activity of wild-type active sites during the rearrangement phase of RNase refolding but <5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI−RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2−8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to “escape” from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi952157n</identifier><identifier>PMID: 8639681</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites - genetics ; Catalysis ; Cattle ; Cysteine - chemistry ; Disulfides - chemistry ; DNA Primers - genetics ; In Vitro Techniques ; Isomerases - chemistry ; Isomerases - genetics ; Isomerases - metabolism ; Kinetics ; Models, Chemical ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Oxidation-Reduction ; Point Mutation ; Protein Disulfide-Isomerases ; Protein Folding ; Rats ; Ribonucleases - chemistry ; Ribonucleases - metabolism</subject><ispartof>Biochemistry (Easton), 1996-02, Vol.35 (6), p.1972-1980</ispartof><rights>Copyright © 1996 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a348t-86c1e79ab9b12720ad537a2718727cca149464dc2d6dcc37eda3d28fb04e17a53</citedby><cites>FETCH-LOGICAL-a348t-86c1e79ab9b12720ad537a2718727cca149464dc2d6dcc37eda3d28fb04e17a53</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/bi952157n$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi952157n$$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/8639681$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Walker, Kenneth W</creatorcontrib><creatorcontrib>Lyles, Michelle M</creatorcontrib><creatorcontrib>Gilbert, Hiram F</creatorcontrib><title>Catalysis of Oxidative Protein Folding by Mutants of Protein Disulfide Isomerase with a Single Active-Site Cysteine</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946−30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 μM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%−50% of the k cat activity of wild-type active sites during the rearrangement phase of RNase refolding but <5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI−RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2−8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to “escape” from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Binding Sites - genetics</subject><subject>Catalysis</subject><subject>Cattle</subject><subject>Cysteine - chemistry</subject><subject>Disulfides - chemistry</subject><subject>DNA Primers - genetics</subject><subject>In Vitro Techniques</subject><subject>Isomerases - chemistry</subject><subject>Isomerases - genetics</subject><subject>Isomerases - metabolism</subject><subject>Kinetics</subject><subject>Models, Chemical</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Oxidation-Reduction</subject><subject>Point Mutation</subject><subject>Protein Disulfide-Isomerases</subject><subject>Protein Folding</subject><subject>Rats</subject><subject>Ribonucleases - chemistry</subject><subject>Ribonucleases - metabolism</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1996</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkEtPwzAQhC0EKqVw4Acg-cKBQ8B2Hk6OVaEPqagVLarExdrYDrikSRUn0Px7UlJ64rRazTe7mkHompJ7Shh9iE3kM-rz7AR1qc-I40WRf4q6hJDAYVFAztGFtetm9Qj3OqgTBm4UhLSL7ABKSGtrLM4TPNsZBaX50nhe5KU2GR7mqTLZO45r_FyVkJW_3J_6aGyVJkZpPLH5RhdgNf425QcGvGhcqcZ9uT_nLEyp8aC2e5e-RGcJpFZfHWYPvQ6floOxM52NJoP-1AHXC0snDCTVPII4iinjjIDyXQ6M05AzLiVQL_ICT0mmAiWly7UCV7EwiYmnKQff7aG79q4scmsLnYhtYTZQ1IISse9NHHtr2JuW3VbxRqsjeSiq0Z1WN02G3VGG4lME3OW-WM4X4m1Fhqvx6EWsGv625UFasc6rImuS_vP3B_YKhOg</recordid><startdate>19960213</startdate><enddate>19960213</enddate><creator>Walker, Kenneth W</creator><creator>Lyles, Michelle M</creator><creator>Gilbert, Hiram 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>19960213</creationdate><title>Catalysis of Oxidative Protein Folding by Mutants of Protein Disulfide Isomerase with a Single Active-Site Cysteine</title><author>Walker, Kenneth W ; Lyles, Michelle M ; Gilbert, Hiram F</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a348t-86c1e79ab9b12720ad537a2718727cca149464dc2d6dcc37eda3d28fb04e17a53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1996</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Binding Sites - genetics</topic><topic>Catalysis</topic><topic>Cattle</topic><topic>Cysteine - chemistry</topic><topic>Disulfides - chemistry</topic><topic>DNA Primers - genetics</topic><topic>In Vitro Techniques</topic><topic>Isomerases - chemistry</topic><topic>Isomerases - genetics</topic><topic>Isomerases - metabolism</topic><topic>Kinetics</topic><topic>Models, Chemical</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Oxidation-Reduction</topic><topic>Point Mutation</topic><topic>Protein Disulfide-Isomerases</topic><topic>Protein Folding</topic><topic>Rats</topic><topic>Ribonucleases - chemistry</topic><topic>Ribonucleases - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Walker, Kenneth W</creatorcontrib><creatorcontrib>Lyles, Michelle M</creatorcontrib><creatorcontrib>Gilbert, Hiram 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>Walker, Kenneth W</au><au>Lyles, Michelle M</au><au>Gilbert, Hiram F</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Catalysis of Oxidative Protein Folding by Mutants of Protein Disulfide Isomerase with a Single Active-Site Cysteine</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1996-02-13</date><risdate>1996</risdate><volume>35</volume><issue>6</issue><spage>1972</spage><epage>1980</epage><pages>1972-1980</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946−30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 μM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%−50% of the k cat activity of wild-type active sites during the rearrangement phase of RNase refolding but <5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI−RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2−8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to “escape” from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>8639681</pmid><doi>10.1021/bi952157n</doi><tpages>9</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0006-2960 |
| ispartof | Biochemistry (Easton), 1996-02, Vol.35 (6), p.1972-1980 |
| issn | 0006-2960 1520-4995 |
| language | eng |
| recordid | cdi_crossref_primary_10_1021_bi952157n |
| source | MEDLINE; American Chemical Society Journals |
| subjects | Amino Acid Sequence Animals Base Sequence Binding Sites - genetics Catalysis Cattle Cysteine - chemistry Disulfides - chemistry DNA Primers - genetics In Vitro Techniques Isomerases - chemistry Isomerases - genetics Isomerases - metabolism Kinetics Models, Chemical Molecular Sequence Data Mutagenesis, Site-Directed Oxidation-Reduction Point Mutation Protein Disulfide-Isomerases Protein Folding Rats Ribonucleases - chemistry Ribonucleases - metabolism |
| title | Catalysis of Oxidative Protein Folding by Mutants of Protein Disulfide Isomerase with a Single Active-Site Cysteine |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-28T08%3A26%3A42IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-istex_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Catalysis%20of%20Oxidative%20Protein%20Folding%20by%20Mutants%20of%20Protein%20Disulfide%20Isomerase%20with%20a%20Single%20Active-Site%20Cysteine&rft.jtitle=Biochemistry%20(Easton)&rft.au=Walker,%20Kenneth%20W&rft.date=1996-02-13&rft.volume=35&rft.issue=6&rft.spage=1972&rft.epage=1980&rft.pages=1972-1980&rft.issn=0006-2960&rft.eissn=1520-4995&rft_id=info:doi/10.1021/bi952157n&rft_dat=%3Cistex_cross%3Eark_67375_TPS_ZW0FWHGR_W%3C/istex_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_id=info:pmid/8639681&rfr_iscdi=true |