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Characterizing lysine acetylation of glucokinase
Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose‐6‐phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on G...
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Published in: | Protein science 2024-01, Vol.33 (1), p.e4845-n/a |
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description | Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose‐6‐phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on GK functions remains largely unknown. In this study, we applied the genetic code expansion strategy to produce site‐specifically acetylated GK variants which naturally exist in cells. Enzyme assays and kinetic analyses showed that lysine acetylation decreases the GK activity, mostly resulting from acetylation of K214 and K216 at the entrance of the active site, which impairs the binding of substrates. We also compared results obtained from the glutamine substitution method and the genetic acetyllysine incorporation approach, showing that glutamine substitution is not always effective for mimicking acetylated lysine. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to determine acetylation and deacetylation mechanisms, which showed that E. coli GK could be acetylated by acetyl‐phosphate without enzymes and deacetylated by CobB deacetylase. |
doi_str_mv | 10.1002/pro.4845 |
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Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on GK functions remains largely unknown. In this study, we applied the genetic code expansion strategy to produce site‐specifically acetylated GK variants which naturally exist in cells. Enzyme assays and kinetic analyses showed that lysine acetylation decreases the GK activity, mostly resulting from acetylation of K214 and K216 at the entrance of the active site, which impairs the binding of substrates. We also compared results obtained from the glutamine substitution method and the genetic acetyllysine incorporation approach, showing that glutamine substitution is not always effective for mimicking acetylated lysine. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to determine acetylation and deacetylation mechanisms, which showed that E. coli GK could be acetylated by acetyl‐phosphate without enzymes and deacetylated by CobB deacetylase.</description><identifier>ISSN: 0961-8368</identifier><identifier>EISSN: 1469-896X</identifier><identifier>DOI: 10.1002/pro.4845</identifier><identifier>PMID: 37996965</identifier><language>eng</language><publisher>Hoboken, USA: John Wiley & Sons, Inc</publisher><subject>Acetylation ; Acetyllysine ; deacetylase ; Deacetylation ; E coli ; Escherichia coli - metabolism ; Genetic code ; genetic code expansion ; Glucokinase ; Glucokinase - genetics ; Glucokinase - metabolism ; Glucose ; Glutamine ; Glutamine - genetics ; Glutamine - metabolism ; Glycolysis ; Lysine ; Lysine - genetics ; lysine acetylation ; Phosphorylation ; Protein Processing, Post-Translational ; Proteomics ; Substitutes ; Substrates</subject><ispartof>Protein science, 2024-01, Vol.33 (1), p.e4845-n/a</ispartof><rights>2023 The Protein Society.</rights><rights>2024 The Protein Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c3105-10427630a606b1e35566bc367010ccf53a95698b8ea31e535edd45e9eec3b7d43</cites><orcidid>0000-0003-0662-1185</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37996965$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Fatema, Nour</creatorcontrib><creatorcontrib>Li, Xinyu</creatorcontrib><creatorcontrib>Gan, Qinglei</creatorcontrib><creatorcontrib>Fan, Chenguang</creatorcontrib><title>Characterizing lysine acetylation of glucokinase</title><title>Protein science</title><addtitle>Protein Sci</addtitle><description>Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose‐6‐phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on GK functions remains largely unknown. In this study, we applied the genetic code expansion strategy to produce site‐specifically acetylated GK variants which naturally exist in cells. Enzyme assays and kinetic analyses showed that lysine acetylation decreases the GK activity, mostly resulting from acetylation of K214 and K216 at the entrance of the active site, which impairs the binding of substrates. We also compared results obtained from the glutamine substitution method and the genetic acetyllysine incorporation approach, showing that glutamine substitution is not always effective for mimicking acetylated lysine. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to determine acetylation and deacetylation mechanisms, which showed that E. coli GK could be acetylated by acetyl‐phosphate without enzymes and deacetylated by CobB deacetylase.</description><subject>Acetylation</subject><subject>Acetyllysine</subject><subject>deacetylase</subject><subject>Deacetylation</subject><subject>E coli</subject><subject>Escherichia coli - metabolism</subject><subject>Genetic code</subject><subject>genetic code expansion</subject><subject>Glucokinase</subject><subject>Glucokinase - genetics</subject><subject>Glucokinase - metabolism</subject><subject>Glucose</subject><subject>Glutamine</subject><subject>Glutamine - genetics</subject><subject>Glutamine - metabolism</subject><subject>Glycolysis</subject><subject>Lysine</subject><subject>Lysine - genetics</subject><subject>lysine acetylation</subject><subject>Phosphorylation</subject><subject>Protein Processing, Post-Translational</subject><subject>Proteomics</subject><subject>Substitutes</subject><subject>Substrates</subject><issn>0961-8368</issn><issn>1469-896X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><recordid>eNp1kF1LwzAUQIMobk7BXyADX3zpTJqPJo8y_AJhIgq-hTS9nZldM5MWqb_ezk0FwafLhcPh3oPQMcETgnF6vgp-wiTjO2hImFCJVOJ5Fw2xEiSRVMgBOohxgTFmJKX7aEAzpYQSfIjw9MUEYxsI7sPV83HVRVfD2Fhouso0ztdjX47nVWv9q6tNhEO0V5oqwtF2jtDT1eXj9Ca5m13fTi_uEksJ5gnBLM0ExUZgkROgnAuRWyoyTLC1JadGcaFkLsFQApxyKArGQQFYmmcFoyN0tvH2v721EBu9dNFCVZkafBt1KhWVlHEie_T0D7rwbaj763SqsCAyFUr9Cm3wMQYo9Sq4pQmdJlivK_a71-uKPXqyFbb5Eoof8DtbDyQb4N1V0P0r0vcPsy_hJ43Meco</recordid><startdate>202401</startdate><enddate>202401</enddate><creator>Fatema, Nour</creator><creator>Li, Xinyu</creator><creator>Gan, Qinglei</creator><creator>Fan, Chenguang</creator><general>John Wiley & Sons, Inc</general><general>Wiley Subscription Services, Inc</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>7QO</scope><scope>7T5</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0003-0662-1185</orcidid></search><sort><creationdate>202401</creationdate><title>Characterizing lysine acetylation of glucokinase</title><author>Fatema, Nour ; Li, Xinyu ; Gan, Qinglei ; Fan, Chenguang</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3105-10427630a606b1e35566bc367010ccf53a95698b8ea31e535edd45e9eec3b7d43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2024</creationdate><topic>Acetylation</topic><topic>Acetyllysine</topic><topic>deacetylase</topic><topic>Deacetylation</topic><topic>E coli</topic><topic>Escherichia coli - metabolism</topic><topic>Genetic code</topic><topic>genetic code expansion</topic><topic>Glucokinase</topic><topic>Glucokinase - genetics</topic><topic>Glucokinase - metabolism</topic><topic>Glucose</topic><topic>Glutamine</topic><topic>Glutamine - genetics</topic><topic>Glutamine - metabolism</topic><topic>Glycolysis</topic><topic>Lysine</topic><topic>Lysine - genetics</topic><topic>lysine acetylation</topic><topic>Phosphorylation</topic><topic>Protein Processing, Post-Translational</topic><topic>Proteomics</topic><topic>Substitutes</topic><topic>Substrates</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Fatema, Nour</creatorcontrib><creatorcontrib>Li, Xinyu</creatorcontrib><creatorcontrib>Gan, Qinglei</creatorcontrib><creatorcontrib>Fan, Chenguang</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Immunology Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Protein science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Fatema, Nour</au><au>Li, Xinyu</au><au>Gan, Qinglei</au><au>Fan, Chenguang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterizing lysine acetylation of glucokinase</atitle><jtitle>Protein science</jtitle><addtitle>Protein Sci</addtitle><date>2024-01</date><risdate>2024</risdate><volume>33</volume><issue>1</issue><spage>e4845</spage><epage>n/a</epage><pages>e4845-n/a</pages><issn>0961-8368</issn><eissn>1469-896X</eissn><abstract>Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose‐6‐phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on GK functions remains largely unknown. In this study, we applied the genetic code expansion strategy to produce site‐specifically acetylated GK variants which naturally exist in cells. Enzyme assays and kinetic analyses showed that lysine acetylation decreases the GK activity, mostly resulting from acetylation of K214 and K216 at the entrance of the active site, which impairs the binding of substrates. We also compared results obtained from the glutamine substitution method and the genetic acetyllysine incorporation approach, showing that glutamine substitution is not always effective for mimicking acetylated lysine. 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subjects | Acetylation Acetyllysine deacetylase Deacetylation E coli Escherichia coli - metabolism Genetic code genetic code expansion Glucokinase Glucokinase - genetics Glucokinase - metabolism Glucose Glutamine Glutamine - genetics Glutamine - metabolism Glycolysis Lysine Lysine - genetics lysine acetylation Phosphorylation Protein Processing, Post-Translational Proteomics Substitutes Substrates |
title | Characterizing lysine acetylation of glucokinase |
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