Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity
Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase f...
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Veröffentlicht in: | The Journal of biological chemistry 1987-07, Vol.262 (21), p.10120-10126 |
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description | Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure. |
doi_str_mv | 10.1016/S0021-9258(18)61086-5 |
format | Article |
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Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity</title><source>Electronic Journals Library</source><source>MEDLINE</source><source>Alma/SFX Local Collection</source><creator>Little, R.M. ; Anderson, P.M.</creator><creatorcontrib>Little, R.M. ; Anderson, P.M.</creatorcontrib><description>Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(18)61086-5</identifier><identifier>PMID: 3301828</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: Elsevier Inc</publisher><subject>Aminohydrolases - metabolism ; Analytical, structural and metabolic biochemistry ; Applied sciences ; Bicarbonates - metabolism ; Biological and medical sciences ; Carbon-Nitrogen Lyases ; Circular Dichroism ; Dithionitrobenzoic Acid - pharmacology ; Enzymes and enzyme inhibitors ; Escherichia coli - enzymology ; Exact sciences and technology ; Fundamental and applied biological sciences. Psychology ; Guanidine ; Guanidines - metabolism ; Hydrolases ; Other techniques and industries ; Polymers ; Protein Conformation ; Protein Denaturation ; Sulfhydryl Compounds - metabolism</subject><ispartof>The Journal of biological chemistry, 1987-07, Vol.262 (21), p.10120-10126</ispartof><rights>1987 © 1987 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>1988 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c494t-dbf9a2d9f8c6f81ed35c80030d06c57afd66ac5de1facfb612fcb841d003e85d3</citedby><cites>FETCH-LOGICAL-c494t-dbf9a2d9f8c6f81ed35c80030d06c57afd66ac5de1facfb612fcb841d003e85d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=7750806$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=7845374$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3301828$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Little, R.M.</creatorcontrib><creatorcontrib>Anderson, P.M.</creatorcontrib><title>Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.</description><subject>Aminohydrolases - metabolism</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Applied sciences</subject><subject>Bicarbonates - metabolism</subject><subject>Biological and medical sciences</subject><subject>Carbon-Nitrogen Lyases</subject><subject>Circular Dichroism</subject><subject>Dithionitrobenzoic Acid - pharmacology</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Escherichia coli - enzymology</subject><subject>Exact sciences and technology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Guanidine</subject><subject>Guanidines - metabolism</subject><subject>Hydrolases</subject><subject>Other techniques and industries</subject><subject>Polymers</subject><subject>Protein Conformation</subject><subject>Protein Denaturation</subject><subject>Sulfhydryl Compounds - metabolism</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1987</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkE1v1DAQhi0EKkvhJ1TKASGQSLGT2HFOCJVPqRKHgsTNcsbjrlE2XmynKHd-OM5utIhTfbE987zz8RJyweglo0y8uaG0YmVXcfmSyVeCUSlK_oBs8qMua85-PCSbE_KYPInxJ82n6dgZOatrymQlN-TPTQoTpCnoodgHv8eQHMbC2wJmPeqIl8V7HPUCJOfH10X476dHUwQ_4CKI02C3swnzEA9xP7hbv8PgoIhrEyzcWIBOephTDmtI7s6l-Sl5ZPUQ8dl6n5PvHz98u_pcXn_99OXq3XUJTdek0vS205XprARhJUNTc5CU1tRQAbzV1gihgRtkVoPtBass9LJhJjMouanPyYtj3bzprwljUjsXAYdBj-inqNo2a7JHGeRHEIKPMaBV--B2OsyKUbW4rw7uq8VaxaQ6uK941l2sDaZ-h-akWu3O-edrXkfQgw16BBdPWCsbXrfNvVjLqaTiH7Z1t9vfLqDqnYct7lQlKpXHy5NWyzJvjxhma-8cBhXB4QhosgSSMt7ds9ZfXKa9rw</recordid><startdate>19870725</startdate><enddate>19870725</enddate><creator>Little, R.M.</creator><creator>Anderson, P.M.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>IQODW</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>19870725</creationdate><title>Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity</title><author>Little, R.M. ; Anderson, P.M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c494t-dbf9a2d9f8c6f81ed35c80030d06c57afd66ac5de1facfb612fcb841d003e85d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1987</creationdate><topic>Aminohydrolases - metabolism</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Applied sciences</topic><topic>Bicarbonates - metabolism</topic><topic>Biological and medical sciences</topic><topic>Carbon-Nitrogen Lyases</topic><topic>Circular Dichroism</topic><topic>Dithionitrobenzoic Acid - pharmacology</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Escherichia coli - enzymology</topic><topic>Exact sciences and technology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Guanidine</topic><topic>Guanidines - metabolism</topic><topic>Hydrolases</topic><topic>Other techniques and industries</topic><topic>Polymers</topic><topic>Protein Conformation</topic><topic>Protein Denaturation</topic><topic>Sulfhydryl Compounds - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Little, R.M.</creatorcontrib><creatorcontrib>Anderson, P.M.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Pascal-Francis</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>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Little, R.M.</au><au>Anderson, P.M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1987-07-25</date><risdate>1987</risdate><volume>262</volume><issue>21</issue><spage>10120</spage><epage>10126</epage><pages>10120-10126</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.</abstract><cop>Bethesda, MD</cop><pub>Elsevier Inc</pub><pmid>3301828</pmid><doi>10.1016/S0021-9258(18)61086-5</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Aminohydrolases - metabolism Analytical, structural and metabolic biochemistry Applied sciences Bicarbonates - metabolism Biological and medical sciences Carbon-Nitrogen Lyases Circular Dichroism Dithionitrobenzoic Acid - pharmacology Enzymes and enzyme inhibitors Escherichia coli - enzymology Exact sciences and technology Fundamental and applied biological sciences. Psychology Guanidine Guanidines - metabolism Hydrolases Other techniques and industries Polymers Protein Conformation Protein Denaturation Sulfhydryl Compounds - metabolism |
title | Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity |
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