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
Hauptverfasser: Steadman, David J, Spencer, H. Trent, Dunlap, R. Bruce, Berger, Sondra H
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container_issue 17
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creator Steadman, David J
Spencer, H. Trent
Dunlap, R. Bruce
Berger, Sondra H
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. 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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. 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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. 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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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