Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase
Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn2+ as the activating metal ion. The enzyme is a monomer and presents 68% identit...
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| Veröffentlicht in: | Journal of Protein Chemistry 2002-08, Vol.21 (6), p.393-400 |
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| creator | Jabalquinto, Ana María Laivenieks, Maris González-Nilo, Fernando D Yévenes, Alejandro Encinas, María Victoria Zeikus, J Gregory Cardemil, Emilio |
| description | Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn2+ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990-994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3-6 orders of magnitude lower values of Vmax/Km, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased Km values for Mn2+ and PEP as compared with wild-type enzyme, suggesting a role of His225 in Mn2+ and PEP binding. From 1.5-1.6 Kcal/mol lower affinity for the 3'(2')-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His225 and Asp263 in nucleotide binding. |
| doi_str_mv | 10.1023/A:1021178432158 |
| format | Article |
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The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990-994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3-6 orders of magnitude lower values of Vmax/Km, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased Km values for Mn2+ and PEP as compared with wild-type enzyme, suggesting a role of His225 in Mn2+ and PEP binding. From 1.5-1.6 Kcal/mol lower affinity for the 3'(2')-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His225 and Asp263 in nucleotide binding.</description><identifier>ISSN: 0277-8033</identifier><identifier>ISSN: 1572-3887</identifier><identifier>EISSN: 1573-4943</identifier><identifier>DOI: 10.1023/A:1021178432158</identifier><identifier>PMID: 12492149</identifier><language>eng</language><publisher>United States: Springer Nature B.V</publisher><subject>Adenosine diphosphate ; Adenosine triphosphate ; Amino acids ; ATP ; Bacteria ; Binding ; Binding Sites ; Carbon dioxide ; Catalysis ; Circular Dichroism ; E coli ; Enzymes ; Kinases ; Kinetics ; Manganese ; Manganese ions ; Metal ions ; Mutagenesis ; Mutagenesis, Site-Directed ; Mutants ; Nucleotides ; Phosphoenolpyruvate Carboxykinase (ATP) - chemistry ; Phosphoenolpyruvate Carboxykinase (ATP) - genetics ; Phosphoenolpyruvate Carboxykinase (ATP) - isolation & purification ; Phosphoenolpyruvate Carboxykinase (ATP) - metabolism ; Proteobacteria - enzymology ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Residues ; Site-directed mutagenesis</subject><ispartof>Journal of Protein Chemistry, 2002-08, Vol.21 (6), p.393-400</ispartof><rights>Plenum Publishing Corporation 2002.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c421t-f279188c6853b9aa3c013a595237f8aa7f060160b786703ed78159b34fa13e6f3</citedby><cites>FETCH-LOGICAL-c421t-f279188c6853b9aa3c013a595237f8aa7f060160b786703ed78159b34fa13e6f3</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/12492149$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jabalquinto, Ana María</creatorcontrib><creatorcontrib>Laivenieks, Maris</creatorcontrib><creatorcontrib>González-Nilo, Fernando D</creatorcontrib><creatorcontrib>Yévenes, Alejandro</creatorcontrib><creatorcontrib>Encinas, María Victoria</creatorcontrib><creatorcontrib>Zeikus, J Gregory</creatorcontrib><creatorcontrib>Cardemil, Emilio</creatorcontrib><title>Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase</title><title>Journal of Protein Chemistry</title><addtitle>J Protein Chem</addtitle><description>Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn2+ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990-994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3-6 orders of magnitude lower values of Vmax/Km, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased Km values for Mn2+ and PEP as compared with wild-type enzyme, suggesting a role of His225 in Mn2+ and PEP binding. From 1.5-1.6 Kcal/mol lower affinity for the 3'(2')-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His225 and Asp263 in nucleotide binding.</description><subject>Adenosine diphosphate</subject><subject>Adenosine triphosphate</subject><subject>Amino acids</subject><subject>ATP</subject><subject>Bacteria</subject><subject>Binding</subject><subject>Binding Sites</subject><subject>Carbon dioxide</subject><subject>Catalysis</subject><subject>Circular Dichroism</subject><subject>E coli</subject><subject>Enzymes</subject><subject>Kinases</subject><subject>Kinetics</subject><subject>Manganese</subject><subject>Manganese ions</subject><subject>Metal ions</subject><subject>Mutagenesis</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutants</subject><subject>Nucleotides</subject><subject>Phosphoenolpyruvate Carboxykinase (ATP) - chemistry</subject><subject>Phosphoenolpyruvate Carboxykinase (ATP) - genetics</subject><subject>Phosphoenolpyruvate Carboxykinase (ATP) - isolation & purification</subject><subject>Phosphoenolpyruvate Carboxykinase (ATP) - metabolism</subject><subject>Proteobacteria - enzymology</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Residues</subject><subject>Site-directed 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Protein Chem</addtitle><date>2002-08-01</date><risdate>2002</risdate><volume>21</volume><issue>6</issue><spage>393</spage><epage>400</epage><pages>393-400</pages><issn>0277-8033</issn><issn>1572-3887</issn><eissn>1573-4943</eissn><abstract>Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn2+ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990-994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3-6 orders of magnitude lower values of Vmax/Km, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased Km values for Mn2+ and PEP as compared with wild-type enzyme, suggesting a role of His225 in Mn2+ and PEP binding. From 1.5-1.6 Kcal/mol lower affinity for the 3'(2')-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His225 and Asp263 in nucleotide binding.</abstract><cop>United States</cop><pub>Springer Nature B.V</pub><pmid>12492149</pmid><doi>10.1023/A:1021178432158</doi><tpages>8</tpages></addata></record> |
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| ispartof | Journal of Protein Chemistry, 2002-08, Vol.21 (6), p.393-400 |
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| subjects | Adenosine diphosphate Adenosine triphosphate Amino acids ATP Bacteria Binding Binding Sites Carbon dioxide Catalysis Circular Dichroism E coli Enzymes Kinases Kinetics Manganese Manganese ions Metal ions Mutagenesis Mutagenesis, Site-Directed Mutants Nucleotides Phosphoenolpyruvate Carboxykinase (ATP) - chemistry Phosphoenolpyruvate Carboxykinase (ATP) - genetics Phosphoenolpyruvate Carboxykinase (ATP) - isolation & purification Phosphoenolpyruvate Carboxykinase (ATP) - metabolism Proteobacteria - enzymology Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Residues Site-directed mutagenesis |
| title | Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase |
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