Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP-dependent glucokinase from Thermococcus litoralis
•Mutations of the HXE and NXXE motifs in TlGK lead to decreased catalytic activity.•The NXXE mutant E308Q is activated by free Mg2+ and increases the KM for MgADP−.•HXE mutants are inhibited by free Mg2+ and increase the KM for glucose.•Contrary to previous hypothesis, HXE is involved in glucose bin...
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| Veröffentlicht in: | FEBS letters 2015-10, Vol.589 (21), p.3271-3276 |
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| creator | Abarca-Lagunas, María José Rivas-Pardo, Jaime Andrés Ramírez-Sarmiento, César A. Guixé, Victoria |
| description | •Mutations of the HXE and NXXE motifs in TlGK lead to decreased catalytic activity.•The NXXE mutant E308Q is activated by free Mg2+ and increases the KM for MgADP−.•HXE mutants are inhibited by free Mg2+ and increase the KM for glucose.•Contrary to previous hypothesis, HXE is involved in glucose binding during ternary complex formation.
The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP− and was activated by free Mg2+. On the other hand, HXE mutants did not affect the KM for MgADP−, were still inhibited by free Mg2+, and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP–AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation. |
| doi_str_mv | 10.1016/j.febslet.2015.09.013 |
| format | Article |
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The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP− and was activated by free Mg2+. On the other hand, HXE mutants did not affect the KM for MgADP−, were still inhibited by free Mg2+, and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP–AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.</description><identifier>ISSN: 0014-5793</identifier><identifier>EISSN: 1873-3468</identifier><identifier>DOI: 10.1016/j.febslet.2015.09.013</identifier><identifier>PMID: 26428088</identifier><language>eng</language><publisher>England: Elsevier B.V</publisher><subject>ADP-dependent kinase ; Amino Acid Motifs ; Archaeal enzyme ; Archaeal Proteins - chemistry ; Archaeal Proteins - genetics ; Archaeal Proteins - metabolism ; Catalytic Domain ; Conserved Sequence ; Divalent metal cation ; Glucokinase ; Glucokinase - chemistry ; Glucokinase - genetics ; Glucokinase - metabolism ; Glucose - metabolism ; Kinetics ; Models, Molecular ; Mutagenesis, Site-Directed ; Protein–ligand binding ; Thermococcus - enzymology ; Thermococcus - genetics</subject><ispartof>FEBS letters, 2015-10, Vol.589 (21), p.3271-3276</ispartof><rights>2015 Federation of European Biochemical Societies</rights><rights>Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c365t-6597679a856425bec4f9ccc0381bf2f6bc8189f10d8d9d24da4825b92746c4d23</citedby><cites>FETCH-LOGICAL-c365t-6597679a856425bec4f9ccc0381bf2f6bc8189f10d8d9d24da4825b92746c4d23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.febslet.2015.09.013$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26428088$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Abarca-Lagunas, María José</creatorcontrib><creatorcontrib>Rivas-Pardo, Jaime Andrés</creatorcontrib><creatorcontrib>Ramírez-Sarmiento, César A.</creatorcontrib><creatorcontrib>Guixé, Victoria</creatorcontrib><title>Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP-dependent glucokinase from Thermococcus litoralis</title><title>FEBS letters</title><addtitle>FEBS Lett</addtitle><description>•Mutations of the HXE and NXXE motifs in TlGK lead to decreased catalytic activity.•The NXXE mutant E308Q is activated by free Mg2+ and increases the KM for MgADP−.•HXE mutants are inhibited by free Mg2+ and increase the KM for glucose.•Contrary to previous hypothesis, HXE is involved in glucose binding during ternary complex formation.
The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP− and was activated by free Mg2+. On the other hand, HXE mutants did not affect the KM for MgADP−, were still inhibited by free Mg2+, and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP–AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.</description><subject>ADP-dependent kinase</subject><subject>Amino Acid Motifs</subject><subject>Archaeal enzyme</subject><subject>Archaeal Proteins - chemistry</subject><subject>Archaeal Proteins - genetics</subject><subject>Archaeal Proteins - metabolism</subject><subject>Catalytic Domain</subject><subject>Conserved Sequence</subject><subject>Divalent metal cation</subject><subject>Glucokinase</subject><subject>Glucokinase - chemistry</subject><subject>Glucokinase - genetics</subject><subject>Glucokinase - metabolism</subject><subject>Glucose - metabolism</subject><subject>Kinetics</subject><subject>Models, Molecular</subject><subject>Mutagenesis, Site-Directed</subject><subject>Protein–ligand binding</subject><subject>Thermococcus - enzymology</subject><subject>Thermococcus - genetics</subject><issn>0014-5793</issn><issn>1873-3468</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc9vFSEQgInR2Nfqn6Dh6GVXWHZZOJmmP6xJox5q0hthYWh5svCE3SYe_c-lfc9ePU1m8s0MfIPQO0paSij_uG0dTCXA0naEDi2RLaHsBdpQMbKG9Vy8RBtCaN8Mo2RH6LiULam5oPI1Oup43wkixAb9OfelgFl8vMPLPWC3xpqkqAPOKUDByT3VTYoF8gNY_PX29gLraPFVjXNavHuGTs-_NxZ2EC3EBd-F1aSfPupSx-Y045t7yHMyyZi14OCXlHXw5Q165XQo8PYQT9CPy4ubs6vm-tvnL2en141hfFgaPsiRj1KLob59mMD0ThpjCBN0cp3jkxFUSEeJFVbarre6F5WT3dhz09uOnaAP-7m7nH6tUBY1-2IgBB0hrUXRkXE-imFkFR32qMmplAxO7bKfdf6tKFGP9tVWHeyrR_uKSFXt1773hxXrNIN97vqnuwKf9gDUjz54yKoYD9GA9bkeQdnk_7PiLxTOmek</recordid><startdate>20151024</startdate><enddate>20151024</enddate><creator>Abarca-Lagunas, María José</creator><creator>Rivas-Pardo, Jaime Andrés</creator><creator>Ramírez-Sarmiento, César A.</creator><creator>Guixé, Victoria</creator><general>Elsevier B.V</general><scope>6I.</scope><scope>AAFTH</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>20151024</creationdate><title>Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP-dependent glucokinase from Thermococcus litoralis</title><author>Abarca-Lagunas, María José ; Rivas-Pardo, Jaime Andrés ; Ramírez-Sarmiento, César A. ; Guixé, Victoria</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c365t-6597679a856425bec4f9ccc0381bf2f6bc8189f10d8d9d24da4825b92746c4d23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>ADP-dependent kinase</topic><topic>Amino Acid Motifs</topic><topic>Archaeal enzyme</topic><topic>Archaeal Proteins - chemistry</topic><topic>Archaeal Proteins - genetics</topic><topic>Archaeal Proteins - metabolism</topic><topic>Catalytic Domain</topic><topic>Conserved Sequence</topic><topic>Divalent metal cation</topic><topic>Glucokinase</topic><topic>Glucokinase - chemistry</topic><topic>Glucokinase - genetics</topic><topic>Glucokinase - metabolism</topic><topic>Glucose - metabolism</topic><topic>Kinetics</topic><topic>Models, Molecular</topic><topic>Mutagenesis, Site-Directed</topic><topic>Protein–ligand binding</topic><topic>Thermococcus - enzymology</topic><topic>Thermococcus - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Abarca-Lagunas, María José</creatorcontrib><creatorcontrib>Rivas-Pardo, Jaime Andrés</creatorcontrib><creatorcontrib>Ramírez-Sarmiento, César A.</creatorcontrib><creatorcontrib>Guixé, Victoria</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</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>FEBS letters</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Abarca-Lagunas, María José</au><au>Rivas-Pardo, Jaime Andrés</au><au>Ramírez-Sarmiento, César A.</au><au>Guixé, Victoria</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP-dependent glucokinase from Thermococcus litoralis</atitle><jtitle>FEBS letters</jtitle><addtitle>FEBS Lett</addtitle><date>2015-10-24</date><risdate>2015</risdate><volume>589</volume><issue>21</issue><spage>3271</spage><epage>3276</epage><pages>3271-3276</pages><issn>0014-5793</issn><eissn>1873-3468</eissn><abstract>•Mutations of the HXE and NXXE motifs in TlGK lead to decreased catalytic activity.•The NXXE mutant E308Q is activated by free Mg2+ and increases the KM for MgADP−.•HXE mutants are inhibited by free Mg2+ and increase the KM for glucose.•Contrary to previous hypothesis, HXE is involved in glucose binding during ternary complex formation.
The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP− and was activated by free Mg2+. On the other hand, HXE mutants did not affect the KM for MgADP−, were still inhibited by free Mg2+, and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP–AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.</abstract><cop>England</cop><pub>Elsevier B.V</pub><pmid>26428088</pmid><doi>10.1016/j.febslet.2015.09.013</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Wiley Free Content; ScienceDirect Journals (5 years ago - present); EZB-FREE-00999 freely available EZB journals; Wiley Online Library All Journals; Alma/SFX Local Collection |
| subjects | ADP-dependent kinase Amino Acid Motifs Archaeal enzyme Archaeal Proteins - chemistry Archaeal Proteins - genetics Archaeal Proteins - metabolism Catalytic Domain Conserved Sequence Divalent metal cation Glucokinase Glucokinase - chemistry Glucokinase - genetics Glucokinase - metabolism Glucose - metabolism Kinetics Models, Molecular Mutagenesis, Site-Directed Protein–ligand binding Thermococcus - enzymology Thermococcus - genetics |
| title | Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP-dependent glucokinase from Thermococcus litoralis |
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