Site-directed mutagenesis at the regulatory site of fructose 6-phosphate-1-kinase from Bacillus stearothermophilus
We have mutated Arg-25, Asp-59 and Arg-211 to alanine; and Asp-59 also to methionine, in fructose 6-phosphate-l-kinase from B. stearothermophilus (designated as RA25, DA59, RA211 and DM59 respectively). All four mutants did not change the affinity of the enzyme for ATP. RA25 has half the affinity fo...
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| Veröffentlicht in: | Biochemical and biophysical research communications 1988-10, Vol.156 (1), p.537-542 |
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| creator | Valdez, B.C. Chang, S.H. Younathan, E.S. |
| description | We have mutated Arg-25, Asp-59 and Arg-211 to alanine; and Asp-59 also to methionine, in fructose 6-phosphate-l-kinase from
B. stearothermophilus (designated as RA25, DA59, RA211 and DM59 respectively). All four mutants did not change the affinity of the enzyme for ATP. RA25 has half the affinity for fructose 6-phosphate and exhibits sigmoidicity with respect to this substrate (Hill # = 2.0). DA59 has the same affinity for phosphoenolpyruvate (PEP) as the wild type whereas DM59 has 3-fold the affinity for this modulator and the inhibition is reversed by GDP. RA25 and RA211 are 100-fold less sensitive to PEP inhibition which is not relieved by GDP. It is concluded that Arg-25 and Arg-211, but not Asp-59, are involved in the direct binding of PEP and GDP. |
| doi_str_mv | 10.1016/S0006-291X(88)80875-1 |
| format | Article |
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B. stearothermophilus (designated as RA25, DA59, RA211 and DM59 respectively). All four mutants did not change the affinity of the enzyme for ATP. RA25 has half the affinity for fructose 6-phosphate and exhibits sigmoidicity with respect to this substrate (Hill # = 2.0). DA59 has the same affinity for phosphoenolpyruvate (PEP) as the wild type whereas DM59 has 3-fold the affinity for this modulator and the inhibition is reversed by GDP. RA25 and RA211 are 100-fold less sensitive to PEP inhibition which is not relieved by GDP. It is concluded that Arg-25 and Arg-211, but not Asp-59, are involved in the direct binding of PEP and GDP.</description><identifier>ISSN: 0006-291X</identifier><identifier>EISSN: 1090-2104</identifier><identifier>DOI: 10.1016/S0006-291X(88)80875-1</identifier><identifier>PMID: 2972287</identifier><identifier>CODEN: BBRCA9</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>Alanine ; Analytical, structural and metabolic biochemistry ; Arginine ; Aspartic Acid ; Bacillus stearothermophilus ; Biological and medical sciences ; Cloning, Molecular ; Enzymes and enzyme inhibitors ; Fundamental and applied biological sciences. Psychology ; Genes ; Genes, Bacterial ; Genes, Regulator ; Geobacillus stearothermophilus - enzymology ; Geobacillus stearothermophilus - genetics ; Kinetics ; Methionine ; Mutation ; Phosphofructokinase-1 - genetics ; Phosphofructokinase-1 - metabolism ; Transferases</subject><ispartof>Biochemical and biophysical research communications, 1988-10, Vol.156 (1), p.537-542</ispartof><rights>1988 Academic Press, Inc.</rights><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c420t-82625608d17fc40fae0cd80984e45dd8abadd82e64c5833e4fbe04153904fbca3</citedby><cites>FETCH-LOGICAL-c420t-82625608d17fc40fae0cd80984e45dd8abadd82e64c5833e4fbe04153904fbca3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/S0006-291X(88)80875-1$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6778498$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2972287$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Valdez, B.C.</creatorcontrib><creatorcontrib>Chang, S.H.</creatorcontrib><creatorcontrib>Younathan, E.S.</creatorcontrib><title>Site-directed mutagenesis at the regulatory site of fructose 6-phosphate-1-kinase from Bacillus stearothermophilus</title><title>Biochemical and biophysical research communications</title><addtitle>Biochem Biophys Res Commun</addtitle><description>We have mutated Arg-25, Asp-59 and Arg-211 to alanine; and Asp-59 also to methionine, in fructose 6-phosphate-l-kinase from
B. stearothermophilus (designated as RA25, DA59, RA211 and DM59 respectively). All four mutants did not change the affinity of the enzyme for ATP. RA25 has half the affinity for fructose 6-phosphate and exhibits sigmoidicity with respect to this substrate (Hill # = 2.0). DA59 has the same affinity for phosphoenolpyruvate (PEP) as the wild type whereas DM59 has 3-fold the affinity for this modulator and the inhibition is reversed by GDP. RA25 and RA211 are 100-fold less sensitive to PEP inhibition which is not relieved by GDP. It is concluded that Arg-25 and Arg-211, but not Asp-59, are involved in the direct binding of PEP and GDP.</description><subject>Alanine</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Arginine</subject><subject>Aspartic Acid</subject><subject>Bacillus stearothermophilus</subject><subject>Biological and medical sciences</subject><subject>Cloning, Molecular</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genes</subject><subject>Genes, Bacterial</subject><subject>Genes, Regulator</subject><subject>Geobacillus stearothermophilus - enzymology</subject><subject>Geobacillus stearothermophilus - genetics</subject><subject>Kinetics</subject><subject>Methionine</subject><subject>Mutation</subject><subject>Phosphofructokinase-1 - genetics</subject><subject>Phosphofructokinase-1 - metabolism</subject><subject>Transferases</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1988</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU2PFCEQhonRrOPqT9iEgzF6aC0YmqZPZt34lWziYTXxRhi62EG7m5aiTfbfy-5M5uoFSNVTL-SBsQsBbwUI_e4GAHQje_HztTFvDJiubcQjthHQQyMFqMdsc0KesmdEvwCEULo_Y2ey76Q03Yblm1iwGWJGX3Dg01rcLc5IkbgrvOyRZ7xdR1dSvuNUWZ4CD3n1JRFy3Sz7RMve1QzR_I6zq8WQ08Q_OB_HcSVOBV1ONShPadnHWnrOngQ3Er447ufsx6eP36--NNffPn-9urxuvJJQGiO1bDWYQXTBKwgOwQ8GeqNQtcNg3M7VVaJWvjXbLaqwQ1Ci3fZQj95tz9mrQ-6S058VqdgpksdxdDOmlaxoheiV1hVsD6DPiShjsEuOk8t3VoC9d20fXNt7kdYY--Daijp3cbxg3U04nKaOcmv_5bHvyLsxZDf7SCdMd51RvanY-wOGVcbfiNmSjzh7PPyKHVL8z0P-AScWndw</recordid><startdate>19881014</startdate><enddate>19881014</enddate><creator>Valdez, B.C.</creator><creator>Chang, S.H.</creator><creator>Younathan, E.S.</creator><general>Elsevier Inc</general><general>Elsevier</general><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>7QL</scope><scope>C1K</scope></search><sort><creationdate>19881014</creationdate><title>Site-directed mutagenesis at the regulatory site of fructose 6-phosphate-1-kinase from Bacillus stearothermophilus</title><author>Valdez, B.C. ; Chang, S.H. ; Younathan, E.S.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c420t-82625608d17fc40fae0cd80984e45dd8abadd82e64c5833e4fbe04153904fbca3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>Alanine</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Arginine</topic><topic>Aspartic Acid</topic><topic>Bacillus stearothermophilus</topic><topic>Biological and medical sciences</topic><topic>Cloning, Molecular</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genes</topic><topic>Genes, Bacterial</topic><topic>Genes, Regulator</topic><topic>Geobacillus stearothermophilus - enzymology</topic><topic>Geobacillus stearothermophilus - genetics</topic><topic>Kinetics</topic><topic>Methionine</topic><topic>Mutation</topic><topic>Phosphofructokinase-1 - genetics</topic><topic>Phosphofructokinase-1 - metabolism</topic><topic>Transferases</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Valdez, B.C.</creatorcontrib><creatorcontrib>Chang, S.H.</creatorcontrib><creatorcontrib>Younathan, E.S.</creatorcontrib><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>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Valdez, B.C.</au><au>Chang, S.H.</au><au>Younathan, E.S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Site-directed mutagenesis at the regulatory site of fructose 6-phosphate-1-kinase from Bacillus stearothermophilus</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>1988-10-14</date><risdate>1988</risdate><volume>156</volume><issue>1</issue><spage>537</spage><epage>542</epage><pages>537-542</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><coden>BBRCA9</coden><abstract>We have mutated Arg-25, Asp-59 and Arg-211 to alanine; and Asp-59 also to methionine, in fructose 6-phosphate-l-kinase from
B. stearothermophilus (designated as RA25, DA59, RA211 and DM59 respectively). All four mutants did not change the affinity of the enzyme for ATP. RA25 has half the affinity for fructose 6-phosphate and exhibits sigmoidicity with respect to this substrate (Hill # = 2.0). DA59 has the same affinity for phosphoenolpyruvate (PEP) as the wild type whereas DM59 has 3-fold the affinity for this modulator and the inhibition is reversed by GDP. RA25 and RA211 are 100-fold less sensitive to PEP inhibition which is not relieved by GDP. It is concluded that Arg-25 and Arg-211, but not Asp-59, are involved in the direct binding of PEP and GDP.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>2972287</pmid><doi>10.1016/S0006-291X(88)80875-1</doi><tpages>6</tpages></addata></record> |
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| subjects | Alanine Analytical, structural and metabolic biochemistry Arginine Aspartic Acid Bacillus stearothermophilus Biological and medical sciences Cloning, Molecular Enzymes and enzyme inhibitors Fundamental and applied biological sciences. Psychology Genes Genes, Bacterial Genes, Regulator Geobacillus stearothermophilus - enzymology Geobacillus stearothermophilus - genetics Kinetics Methionine Mutation Phosphofructokinase-1 - genetics Phosphofructokinase-1 - metabolism Transferases |
| title | Site-directed mutagenesis at the regulatory site of fructose 6-phosphate-1-kinase from Bacillus stearothermophilus |
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