States and Functions of Tyrosine Residues in Escherichia coli Asparaginase II
The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site‐directed mutagenesis, chemical modification of wild‐type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue...
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| Veröffentlicht in: | European journal of biochemistry 1994-09, Vol.224 (2), p.533-540 |
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| creator | Derst, Christian Wehner, Andreas Specht, Volker Röhm, Klaus‐Heinrich |
| description | The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site‐directed mutagenesis, chemical modification of wild‐type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active‐site residue Y25 has a normal pKa while the C‐terminal Y326 is unusually acidic. 1H‐NMR signals of a peculiar ligand‐sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14–27) which shields the active site during catalysis is highly flexible in the free enzyme. |
| doi_str_mv | 10.1111/j.1432-1033.1994.00533.x |
| format | Article |
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While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active‐site residue Y25 has a normal pKa while the C‐terminal Y326 is unusually acidic. 1H‐NMR signals of a peculiar ligand‐sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14–27) which shields the active site during catalysis is highly flexible in the free enzyme.</description><identifier>ISSN: 0014-2956</identifier><identifier>EISSN: 1432-1033</identifier><identifier>DOI: 10.1111/j.1432-1033.1994.00533.x</identifier><identifier>PMID: 7925369</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Science Ltd</publisher><subject>Asparaginase - biosynthesis ; Asparaginase - chemistry ; Asparaginase - metabolism ; Base Sequence ; Bromosuccinimide ; DNA Primers ; Escherichia coli ; Escherichia coli - enzymology ; Guanidine ; Guanidines ; Hydrogen-Ion Concentration ; Macromolecular Substances ; Magnetic Resonance Spectroscopy ; Models, Structural ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Protein Denaturation ; Protein Structure, Secondary ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - metabolism ; Thermodynamics ; Tyrosine</subject><ispartof>European journal of biochemistry, 1994-09, Vol.224 (2), p.533-540</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4453-a095a6c5ef8a916692d5d521669540b66deedd4dbcd46931ade652bd456ae3923</citedby><cites>FETCH-LOGICAL-c4453-a095a6c5ef8a916692d5d521669540b66deedd4dbcd46931ade652bd456ae3923</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/7925369$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Derst, Christian</creatorcontrib><creatorcontrib>Wehner, Andreas</creatorcontrib><creatorcontrib>Specht, Volker</creatorcontrib><creatorcontrib>Röhm, Klaus‐Heinrich</creatorcontrib><title>States and Functions of Tyrosine Residues in Escherichia coli Asparaginase II</title><title>European journal of biochemistry</title><addtitle>Eur J Biochem</addtitle><description>The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site‐directed mutagenesis, chemical modification of wild‐type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active‐site residue Y25 has a normal pKa while the C‐terminal Y326 is unusually acidic. 1H‐NMR signals of a peculiar ligand‐sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14–27) which shields the active site during catalysis is highly flexible in the free enzyme.</description><subject>Asparaginase - biosynthesis</subject><subject>Asparaginase - chemistry</subject><subject>Asparaginase - metabolism</subject><subject>Base Sequence</subject><subject>Bromosuccinimide</subject><subject>DNA Primers</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Guanidine</subject><subject>Guanidines</subject><subject>Hydrogen-Ion Concentration</subject><subject>Macromolecular Substances</subject><subject>Magnetic Resonance Spectroscopy</subject><subject>Models, Structural</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Protein Denaturation</subject><subject>Protein Structure, Secondary</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - metabolism</subject><subject>Thermodynamics</subject><subject>Tyrosine</subject><issn>0014-2956</issn><issn>1432-1033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1994</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkM1OAjEUhRujQUQfwaQrdzO20x_owgUSUBKMieC66bQXKRlmcMpEeHs7QljbTU9yzj3t_RDClKQ0nsd1SjnLEkoYS6lSPCVERLm_QN2zcYm6hFCeZErIa3QTwpoQIpXsd1CnrzLBpOqit_nO7CBgUzo8aUq781UZcLXEi0NdBV8C_oDgXRMjvsTjYFdQe7vyBtuq8HgYtqY2X740AfB0eouulqYIcHe6e-hzMl6MXpPZ-8t0NJwllnPBEkOUMNIKWA6MolKqzAknslYJTnIpHYBz3OXWcakYNQ6kyHLHhTTAVMZ66OHYu62r7_i3nd74YKEoTAlVE3Rs4jzuHoODY9DGbUINS72t_cbUB02JbknqtW6B6RaYbknqP5J6H0fvT280-QbcefCELvpPR__HF3D4d6-ejJ_nUbFfHXCBWA</recordid><startdate>199409</startdate><enddate>199409</enddate><creator>Derst, Christian</creator><creator>Wehner, Andreas</creator><creator>Specht, Volker</creator><creator>Röhm, Klaus‐Heinrich</creator><general>Blackwell Science Ltd</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><scope>7QL</scope><scope>7TM</scope><scope>C1K</scope></search><sort><creationdate>199409</creationdate><title>States and Functions of Tyrosine Residues in Escherichia coli Asparaginase II</title><author>Derst, Christian ; Wehner, Andreas ; Specht, Volker ; Röhm, Klaus‐Heinrich</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4453-a095a6c5ef8a916692d5d521669540b66deedd4dbcd46931ade652bd456ae3923</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1994</creationdate><topic>Asparaginase - biosynthesis</topic><topic>Asparaginase - chemistry</topic><topic>Asparaginase - metabolism</topic><topic>Base Sequence</topic><topic>Bromosuccinimide</topic><topic>DNA Primers</topic><topic>Escherichia coli</topic><topic>Escherichia coli - enzymology</topic><topic>Guanidine</topic><topic>Guanidines</topic><topic>Hydrogen-Ion Concentration</topic><topic>Macromolecular Substances</topic><topic>Magnetic Resonance Spectroscopy</topic><topic>Models, Structural</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Protein Denaturation</topic><topic>Protein Structure, Secondary</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - metabolism</topic><topic>Thermodynamics</topic><topic>Tyrosine</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Derst, Christian</creatorcontrib><creatorcontrib>Wehner, Andreas</creatorcontrib><creatorcontrib>Specht, Volker</creatorcontrib><creatorcontrib>Röhm, Klaus‐Heinrich</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><jtitle>European journal of biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Derst, Christian</au><au>Wehner, Andreas</au><au>Specht, Volker</au><au>Röhm, Klaus‐Heinrich</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>States and Functions of Tyrosine Residues in Escherichia coli Asparaginase II</atitle><jtitle>European journal of biochemistry</jtitle><addtitle>Eur J Biochem</addtitle><date>1994-09</date><risdate>1994</risdate><volume>224</volume><issue>2</issue><spage>533</spage><epage>540</epage><pages>533-540</pages><issn>0014-2956</issn><eissn>1432-1033</eissn><abstract>The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site‐directed mutagenesis, chemical modification of wild‐type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active‐site residue Y25 has a normal pKa while the C‐terminal Y326 is unusually acidic. 1H‐NMR signals of a peculiar ligand‐sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14–27) which shields the active site during catalysis is highly flexible in the free enzyme.</abstract><cop>Oxford, UK</cop><pub>Blackwell Science Ltd</pub><pmid>7925369</pmid><doi>10.1111/j.1432-1033.1994.00533.x</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
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| ispartof | European journal of biochemistry, 1994-09, Vol.224 (2), p.533-540 |
| issn | 0014-2956 1432-1033 |
| language | eng |
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| subjects | Asparaginase - biosynthesis Asparaginase - chemistry Asparaginase - metabolism Base Sequence Bromosuccinimide DNA Primers Escherichia coli Escherichia coli - enzymology Guanidine Guanidines Hydrogen-Ion Concentration Macromolecular Substances Magnetic Resonance Spectroscopy Models, Structural Molecular Sequence Data Mutagenesis, Site-Directed Protein Denaturation Protein Structure, Secondary Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - metabolism Thermodynamics Tyrosine |
| title | States and Functions of Tyrosine Residues in Escherichia coli Asparaginase II |
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