Substitution at Residue 214 of Human Thymidylate Synthase Alters Nucleotide Binding and Isomerization of Ligand−Protein Complexes
Based on crystal structures of bacterial thymidylate synthases (TS), a glutamine corresponding to residue 214 in human TS (hTS) is located in a region that is postulated to be critical for conformational changes that occur upon ligand binding. Previous steady-state kinetic studies indicated that rep...
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Veröffentlicht in: | Biochemistry (Easton) 1999-04, Vol.38 (17), p.5582-5587 |
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description | Based on crystal structures of bacterial thymidylate synthases (TS), a glutamine corresponding to residue 214 in human TS (hTS) is located in a region that is postulated to be critical for conformational changes that occur upon ligand binding. Previous steady-state kinetic studies indicated that replacement of glutamine at position 214 (Gln214) of hTS by other residues results in a decrease in nucleotide binding and catalysis, with only minor effects on folate binding (D. J. Steadman et al. (1998) Biochemistry 37, 7089−7095). The data suggested that Gln214 maintains the enzyme in a conformation that facilitates nucleotide binding. In the present study, transient-state kinetic analysis was utilized to determine rate constants that govern specific steps along the catalytic pathway of hTS, which provides the first detailed kinetic mechanism for hTS. Analysis of the reaction mechanisms of mutant TSs revealed that substitution at position 214 significantly affects nucleotide binding and the rate of chemical conversion of bound substrates to products, which is consistent with the results of steady-state kinetic analysis. Furthermore, it is shown that substitution at position 214 affects the rate of isomerization, presumably from an open to a closed form of the enzyme−substrate complex. Although the affinity of the initial binding of CH2H4folate is not substantially affected, K iso, the ratio of the forward rate of isomerization (k iso) to the reverse rate of isomerization (k r,iso), is 2−6-fold lower for the mutants at position 214 compared to Q214, with the greatest effects on k iso. In addition, the binding of the folate analogue, CB3717, to dUMP binary complexes of mutant enzymes was characterized by a slow isomerization phase that was not detected in binding studies utilizing wild-type hTS. The data are consistent with the hypothesis that Gln214 is located at a structurally critical region of the enzyme. |
doi_str_mv | 10.1021/bi982910n |
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Trent ; Dunlap, R. Bruce ; Berger, Sondra H</creator><creatorcontrib>Steadman, David J ; Spencer, H. Trent ; Dunlap, R. Bruce ; Berger, Sondra H</creatorcontrib><description>Based on crystal structures of bacterial thymidylate synthases (TS), a glutamine corresponding to residue 214 in human TS (hTS) is located in a region that is postulated to be critical for conformational changes that occur upon ligand binding. Previous steady-state kinetic studies indicated that replacement of glutamine at position 214 (Gln214) of hTS by other residues results in a decrease in nucleotide binding and catalysis, with only minor effects on folate binding (D. J. Steadman et al. (1998) Biochemistry 37, 7089−7095). The data suggested that Gln214 maintains the enzyme in a conformation that facilitates nucleotide binding. In the present study, transient-state kinetic analysis was utilized to determine rate constants that govern specific steps along the catalytic pathway of hTS, which provides the first detailed kinetic mechanism for hTS. Analysis of the reaction mechanisms of mutant TSs revealed that substitution at position 214 significantly affects nucleotide binding and the rate of chemical conversion of bound substrates to products, which is consistent with the results of steady-state kinetic analysis. Furthermore, it is shown that substitution at position 214 affects the rate of isomerization, presumably from an open to a closed form of the enzyme−substrate complex. Although the affinity of the initial binding of CH2H4folate is not substantially affected, K iso, the ratio of the forward rate of isomerization (k iso) to the reverse rate of isomerization (k r,iso), is 2−6-fold lower for the mutants at position 214 compared to Q214, with the greatest effects on k iso. In addition, the binding of the folate analogue, CB3717, to dUMP binary complexes of mutant enzymes was characterized by a slow isomerization phase that was not detected in binding studies utilizing wild-type hTS. The data are consistent with the hypothesis that Gln214 is located at a structurally critical region of the enzyme.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi982910n</identifier><identifier>PMID: 10220346</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amino Acid Substitution - genetics ; Binding Sites - genetics ; Deoxyuracil Nucleotides - metabolism ; Enzyme Activation ; Glutamine - genetics ; Humans ; Isoenzymes - genetics ; Isoenzymes - metabolism ; Kinetics ; Ligands ; Macromolecular Substances ; Mutagenesis, Site-Directed ; Spectrometry, Fluorescence ; Tetrahydrofolates - metabolism ; Thermodynamics ; Thymidine Monophosphate - metabolism ; Thymidylate Synthase - genetics ; Thymidylate Synthase - metabolism</subject><ispartof>Biochemistry (Easton), 1999-04, Vol.38 (17), p.5582-5587</ispartof><rights>Copyright © 1999 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a349t-f023da059516b132521efecf49bccb4df0f8ac05c4ce8c5b91f4f2295acb76e83</citedby><cites>FETCH-LOGICAL-a349t-f023da059516b132521efecf49bccb4df0f8ac05c4ce8c5b91f4f2295acb76e83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi982910n$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi982910n$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>315,782,786,2767,27083,27931,27932,56745,56795</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10220346$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Steadman, David J</creatorcontrib><creatorcontrib>Spencer, H. Trent</creatorcontrib><creatorcontrib>Dunlap, R. Bruce</creatorcontrib><creatorcontrib>Berger, Sondra H</creatorcontrib><title>Substitution at Residue 214 of Human Thymidylate Synthase Alters Nucleotide Binding and Isomerization of Ligand−Protein Complexes</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Based on crystal structures of bacterial thymidylate synthases (TS), a glutamine corresponding to residue 214 in human TS (hTS) is located in a region that is postulated to be critical for conformational changes that occur upon ligand binding. Previous steady-state kinetic studies indicated that replacement of glutamine at position 214 (Gln214) of hTS by other residues results in a decrease in nucleotide binding and catalysis, with only minor effects on folate binding (D. J. Steadman et al. (1998) Biochemistry 37, 7089−7095). The data suggested that Gln214 maintains the enzyme in a conformation that facilitates nucleotide binding. In the present study, transient-state kinetic analysis was utilized to determine rate constants that govern specific steps along the catalytic pathway of hTS, which provides the first detailed kinetic mechanism for hTS. Analysis of the reaction mechanisms of mutant TSs revealed that substitution at position 214 significantly affects nucleotide binding and the rate of chemical conversion of bound substrates to products, which is consistent with the results of steady-state kinetic analysis. Furthermore, it is shown that substitution at position 214 affects the rate of isomerization, presumably from an open to a closed form of the enzyme−substrate complex. Although the affinity of the initial binding of CH2H4folate is not substantially affected, K iso, the ratio of the forward rate of isomerization (k iso) to the reverse rate of isomerization (k r,iso), is 2−6-fold lower for the mutants at position 214 compared to Q214, with the greatest effects on k iso. In addition, the binding of the folate analogue, CB3717, to dUMP binary complexes of mutant enzymes was characterized by a slow isomerization phase that was not detected in binding studies utilizing wild-type hTS. The data are consistent with the hypothesis that Gln214 is located at a structurally critical region of the enzyme.</description><subject>Amino Acid Substitution - genetics</subject><subject>Binding Sites - genetics</subject><subject>Deoxyuracil Nucleotides - metabolism</subject><subject>Enzyme Activation</subject><subject>Glutamine - genetics</subject><subject>Humans</subject><subject>Isoenzymes - genetics</subject><subject>Isoenzymes - metabolism</subject><subject>Kinetics</subject><subject>Ligands</subject><subject>Macromolecular Substances</subject><subject>Mutagenesis, Site-Directed</subject><subject>Spectrometry, Fluorescence</subject><subject>Tetrahydrofolates - metabolism</subject><subject>Thermodynamics</subject><subject>Thymidine Monophosphate - metabolism</subject><subject>Thymidylate Synthase - genetics</subject><subject>Thymidylate Synthase - metabolism</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkM9u1DAQhy0EokvhwAsgX0DiEGo7TrI-dlfAVlrRFbuoR8txxq1LYm_9R-py5cKZR-yTNJCq4sBpNDPffCP9EHpNyQdKGD1prZgzQYl7gma0YqTgQlRP0YwQUhdM1OQIvYjxemw5afhzdDReMVLyeoZ-bnMbk005We-wSvgrRNtlwIxy7A1e5UE5vLs6DLY79CoB3h5culIR8GmfIET8JesefLId4IV1nXWXWLkOn0U_QLA_1F_xaFrby3F-9-v3JvgE1uGlH_Y93EJ8iZ4Z1Ud49VCP0bdPH3fLVbE-_3y2PF0XquQiFYawslOkEhWtW1qyilEwoA0XrdYt7wwxc6VJpbmGua5aQQ03jIlK6bapYV4eo3eTdx_8TYaY5GCjhr5XDnyOkjasLimlI_h-AnXwMQYwch_soMJBUiL_JC4fEx_ZNw_S3A7Q_UNOEY9AMQE2Jrh93KvwXdZN2VRyt9nKxUZcbJcXC7ka-bcTr3SU1z4HN2byn8f3fHiaLA</recordid><startdate>19990427</startdate><enddate>19990427</enddate><creator>Steadman, David J</creator><creator>Spencer, H. Trent</creator><creator>Dunlap, R. Bruce</creator><creator>Berger, Sondra H</creator><general>American Chemical Society</general><scope>BSCLL</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>7TM</scope></search><sort><creationdate>19990427</creationdate><title>Substitution at Residue 214 of Human Thymidylate Synthase Alters Nucleotide Binding and Isomerization of Ligand−Protein Complexes</title><author>Steadman, David J ; Spencer, H. Trent ; Dunlap, R. Bruce ; Berger, Sondra H</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a349t-f023da059516b132521efecf49bccb4df0f8ac05c4ce8c5b91f4f2295acb76e83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Amino Acid Substitution - genetics</topic><topic>Binding Sites - genetics</topic><topic>Deoxyuracil Nucleotides - metabolism</topic><topic>Enzyme Activation</topic><topic>Glutamine - genetics</topic><topic>Humans</topic><topic>Isoenzymes - genetics</topic><topic>Isoenzymes - metabolism</topic><topic>Kinetics</topic><topic>Ligands</topic><topic>Macromolecular Substances</topic><topic>Mutagenesis, Site-Directed</topic><topic>Spectrometry, Fluorescence</topic><topic>Tetrahydrofolates - metabolism</topic><topic>Thermodynamics</topic><topic>Thymidine Monophosphate - metabolism</topic><topic>Thymidylate Synthase - genetics</topic><topic>Thymidylate Synthase - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Steadman, David J</creatorcontrib><creatorcontrib>Spencer, H. Trent</creatorcontrib><creatorcontrib>Dunlap, R. Bruce</creatorcontrib><creatorcontrib>Berger, Sondra H</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Steadman, David J</au><au>Spencer, H. Trent</au><au>Dunlap, R. Bruce</au><au>Berger, Sondra H</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Substitution at Residue 214 of Human Thymidylate Synthase Alters Nucleotide Binding and Isomerization of Ligand−Protein Complexes</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1999-04-27</date><risdate>1999</risdate><volume>38</volume><issue>17</issue><spage>5582</spage><epage>5587</epage><pages>5582-5587</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Based on crystal structures of bacterial thymidylate synthases (TS), a glutamine corresponding to residue 214 in human TS (hTS) is located in a region that is postulated to be critical for conformational changes that occur upon ligand binding. Previous steady-state kinetic studies indicated that replacement of glutamine at position 214 (Gln214) of hTS by other residues results in a decrease in nucleotide binding and catalysis, with only minor effects on folate binding (D. J. Steadman et al. (1998) Biochemistry 37, 7089−7095). The data suggested that Gln214 maintains the enzyme in a conformation that facilitates nucleotide binding. In the present study, transient-state kinetic analysis was utilized to determine rate constants that govern specific steps along the catalytic pathway of hTS, which provides the first detailed kinetic mechanism for hTS. Analysis of the reaction mechanisms of mutant TSs revealed that substitution at position 214 significantly affects nucleotide binding and the rate of chemical conversion of bound substrates to products, which is consistent with the results of steady-state kinetic analysis. Furthermore, it is shown that substitution at position 214 affects the rate of isomerization, presumably from an open to a closed form of the enzyme−substrate complex. Although the affinity of the initial binding of CH2H4folate is not substantially affected, K iso, the ratio of the forward rate of isomerization (k iso) to the reverse rate of isomerization (k r,iso), is 2−6-fold lower for the mutants at position 214 compared to Q214, with the greatest effects on k iso. In addition, the binding of the folate analogue, CB3717, to dUMP binary complexes of mutant enzymes was characterized by a slow isomerization phase that was not detected in binding studies utilizing wild-type hTS. The data are consistent with the hypothesis that Gln214 is located at a structurally critical region of the enzyme.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>10220346</pmid><doi>10.1021/bi982910n</doi><tpages>6</tpages></addata></record> |
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subjects | Amino Acid Substitution - genetics Binding Sites - genetics Deoxyuracil Nucleotides - metabolism Enzyme Activation Glutamine - genetics Humans Isoenzymes - genetics Isoenzymes - metabolism Kinetics Ligands Macromolecular Substances Mutagenesis, Site-Directed Spectrometry, Fluorescence Tetrahydrofolates - metabolism Thermodynamics Thymidine Monophosphate - metabolism Thymidylate Synthase - genetics Thymidylate Synthase - metabolism |
title | Substitution at Residue 214 of Human Thymidylate Synthase Alters Nucleotide Binding and Isomerization of Ligand−Protein Complexes |
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