Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III
Site-directed mutants of human carbonic anhydrase III were used to examine the role of Thr-199 and its interaction with Phe-198 in the catalyzed hydration of CO2. Threonine-199 is a hydrogen bond acceptor for the zinc-bound water, and Phe-198 forms part of the hydrophobic side of the active-site cav...
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| Veröffentlicht in: | Biochemistry (Easton) 1993-08, Vol.32 (31), p.7861-7865 |
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| container_issue | 31 |
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| container_title | Biochemistry (Easton) |
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| creator | Chen, Xian Tu, Chingkuang LoGrasso, Philip V Laipis, Philip J Silverman, David N |
| description | Site-directed mutants of human carbonic anhydrase III were used to examine the role of Thr-199 and its interaction with Phe-198 in the catalyzed hydration of CO2. Threonine-199 is a hydrogen bond acceptor for the zinc-bound water, and Phe-198 forms part of the hydrophobic side of the active-site cavity of carbonic anhydrase III. Catalytic activity for a total of five single and double mutants at residues 198 and 199 was determined by stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry. The replacement Thr-199-->Ala resulted in a 4-fold decrease in the kcat/Km for hydration of CO2. We tested the hypothesis that the 25-fold increase in the kcat/Km for hydration of CO2 accompanying the replacement Phe-198-->Leu in isozyme III is caused by changes in the interaction of Thr-199 with the zinc-bound water or the transition state for catalysis. Comparison of hydration of CO2 by the single and double mutants of isozyme III containing the replacements Thr-199-->Ala and Phe-198-->Leu was consistent with an interaction between these two sites. |
| doi_str_mv | 10.1021/bi00082a004 |
| format | Article |
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Threonine-199 is a hydrogen bond acceptor for the zinc-bound water, and Phe-198 forms part of the hydrophobic side of the active-site cavity of carbonic anhydrase III. Catalytic activity for a total of five single and double mutants at residues 198 and 199 was determined by stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry. The replacement Thr-199-->Ala resulted in a 4-fold decrease in the kcat/Km for hydration of CO2. We tested the hypothesis that the 25-fold increase in the kcat/Km for hydration of CO2 accompanying the replacement Phe-198-->Leu in isozyme III is caused by changes in the interaction of Thr-199 with the zinc-bound water or the transition state for catalysis. Comparison of hydration of CO2 by the single and double mutants of isozyme III containing the replacements Thr-199-->Ala and Phe-198-->Leu was consistent with an interaction between these two sites.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi00082a004</identifier><identifier>PMID: 8347590</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>Amino Acid Sequence ; Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; Carbonic Anhydrases - chemistry ; Carbonic Anhydrases - metabolism ; Catalysis ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; Humans ; Kinetics ; Lyases ; Models, Chemical ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Oxygen Isotopes ; Phenylalanine ; Threonine</subject><ispartof>Biochemistry (Easton), 1993-08, Vol.32 (31), p.7861-7865</ispartof><rights>1993 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a414t-59ee583291ac9d4511f93297af8f8b6210b6905b5c566ff2a9419dd23bcb25323</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi00082a004$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi00082a004$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2763,27075,27923,27924,56737,56787</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4863686$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8347590$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Chen, Xian</creatorcontrib><creatorcontrib>Tu, Chingkuang</creatorcontrib><creatorcontrib>LoGrasso, Philip V</creatorcontrib><creatorcontrib>Laipis, Philip J</creatorcontrib><creatorcontrib>Silverman, David N</creatorcontrib><title>Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Site-directed mutants of human carbonic anhydrase III were used to examine the role of Thr-199 and its interaction with Phe-198 in the catalyzed hydration of CO2. Threonine-199 is a hydrogen bond acceptor for the zinc-bound water, and Phe-198 forms part of the hydrophobic side of the active-site cavity of carbonic anhydrase III. Catalytic activity for a total of five single and double mutants at residues 198 and 199 was determined by stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry. The replacement Thr-199-->Ala resulted in a 4-fold decrease in the kcat/Km for hydration of CO2. We tested the hypothesis that the 25-fold increase in the kcat/Km for hydration of CO2 accompanying the replacement Phe-198-->Leu in isozyme III is caused by changes in the interaction of Thr-199 with the zinc-bound water or the transition state for catalysis. Comparison of hydration of CO2 by the single and double mutants of isozyme III containing the replacements Thr-199-->Ala and Phe-198-->Leu was consistent with an interaction between these two sites.</description><subject>Amino Acid Sequence</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>Carbonic Anhydrases - chemistry</subject><subject>Carbonic Anhydrases - metabolism</subject><subject>Catalysis</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Lyases</subject><subject>Models, Chemical</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Oxygen Isotopes</subject><subject>Phenylalanine</subject><subject>Threonine</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkM-L1DAUx4Mo6-zoybPQg-hBuuZ3m6MMuhZXFBzBW3hJE6Zrm45JC_a_36xThj3s4fF43-_nPZIvQq8IviKYkg-mwxjXFDDmT9CGCIpLrpR4ijZZlyVVEj9Hlynd5pHjil-gi5rxSii8QUMTJhfBTt0YCght0QXfzy5YV4y-OB5cWHroIXTBlUTV_5HpEN24KqrIexYm6JfUpcIsxWEe4F6KJjM2LxyWNkJyRdM0L9AzD31yL9e-Rb8-f9rvvpQ336-b3cebEjjhUymUc6JmVBGwquWCEK_yVIGvfW0kJdhIhYURVkjpPQXFiWpbyow1VDDKtujt6e4xjn9nlyY9dMm6Pn_EjXPSRMqasIxu0fsTaOOYUnReH2M3QFw0wfo-XP0g3Ey_Xs_OZnDtmV3TzP6b1YdkofcRgu3SGeO1ZDLXFpUnrEuT-3e2If7RsmKV0PsfPzVh-99fFdnpb5l_d-LBJn07zjHk7B594B3LmZuG</recordid><startdate>19930810</startdate><enddate>19930810</enddate><creator>Chen, Xian</creator><creator>Tu, Chingkuang</creator><creator>LoGrasso, Philip V</creator><creator>Laipis, Philip J</creator><creator>Silverman, David N</creator><general>American Chemical Society</general><scope>BSCLL</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>7TM</scope></search><sort><creationdate>19930810</creationdate><title>Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III</title><author>Chen, Xian ; Tu, Chingkuang ; LoGrasso, Philip V ; Laipis, Philip J ; Silverman, David N</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a414t-59ee583291ac9d4511f93297af8f8b6210b6905b5c566ff2a9419dd23bcb25323</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Amino Acid Sequence</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>Carbonic Anhydrases - chemistry</topic><topic>Carbonic Anhydrases - metabolism</topic><topic>Catalysis</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Lyases</topic><topic>Models, Chemical</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Oxygen Isotopes</topic><topic>Phenylalanine</topic><topic>Threonine</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chen, Xian</creatorcontrib><creatorcontrib>Tu, Chingkuang</creatorcontrib><creatorcontrib>LoGrasso, Philip V</creatorcontrib><creatorcontrib>Laipis, Philip J</creatorcontrib><creatorcontrib>Silverman, David N</creatorcontrib><collection>Istex</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>Nucleic Acids Abstracts</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chen, Xian</au><au>Tu, Chingkuang</au><au>LoGrasso, Philip V</au><au>Laipis, Philip J</au><au>Silverman, David N</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1993-08-10</date><risdate>1993</risdate><volume>32</volume><issue>31</issue><spage>7861</spage><epage>7865</epage><pages>7861-7865</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Site-directed mutants of human carbonic anhydrase III were used to examine the role of Thr-199 and its interaction with Phe-198 in the catalyzed hydration of CO2. Threonine-199 is a hydrogen bond acceptor for the zinc-bound water, and Phe-198 forms part of the hydrophobic side of the active-site cavity of carbonic anhydrase III. Catalytic activity for a total of five single and double mutants at residues 198 and 199 was determined by stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry. The replacement Thr-199-->Ala resulted in a 4-fold decrease in the kcat/Km for hydration of CO2. We tested the hypothesis that the 25-fold increase in the kcat/Km for hydration of CO2 accompanying the replacement Phe-198-->Leu in isozyme III is caused by changes in the interaction of Thr-199 with the zinc-bound water or the transition state for catalysis. Comparison of hydration of CO2 by the single and double mutants of isozyme III containing the replacements Thr-199-->Ala and Phe-198-->Leu was consistent with an interaction between these two sites.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>8347590</pmid><doi>10.1021/bi00082a004</doi><tpages>5</tpages></addata></record> |
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| subjects | Amino Acid Sequence Analytical, structural and metabolic biochemistry Biological and medical sciences Carbonic Anhydrases - chemistry Carbonic Anhydrases - metabolism Catalysis Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Humans Kinetics Lyases Models, Chemical Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Oxygen Isotopes Phenylalanine Threonine |
| title | Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III |
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