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==INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS== | ==INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS== | ||
<StructureSection load='3drc' size='340' side='right' caption='[[3drc]], [[Resolution|resolution]] 1.90Å' scene=''> | <StructureSection load='3drc' size='340' side='right' caption='[[3drc]], [[Resolution|resolution]] 1.90Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>[[3drc]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/ | <table><tr><td colspan='2'>[[3drc]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/"bacillus_coli"_migula_1895 "bacillus coli" migula 1895]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3DRC OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3DRC FirstGlance]. <br> | ||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=MTX:METHOTREXATE'>MTX</scene></td></tr> | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=MTX:METHOTREXATE'>MTX</scene></td></tr> | ||
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Dihydrofolate_reductase Dihydrofolate reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.5.1.3 1.5.1.3] </span></td></tr> | <tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Dihydrofolate_reductase Dihydrofolate reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.5.1.3 1.5.1.3] </span></td></tr> | ||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3drc FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3drc OCA], [http://www.rcsb.org/pdb/explore.do?structureId=3drc RCSB], [http://www.ebi.ac.uk/pdbsum/3drc PDBsum]</span></td></tr> | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3drc FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3drc OCA], [http://pdbe.org/3drc PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=3drc RCSB], [http://www.ebi.ac.uk/pdbsum/3drc PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=3drc ProSAT]</span></td></tr> | ||
</table> | </table> | ||
== Function == | == Function == | ||
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<text>to colour the structure by Evolutionary Conservation</text> | <text>to colour the structure by Evolutionary Conservation</text> | ||
</jmolCheckbox> | </jmolCheckbox> | ||
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/ | </jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=3drc ConSurf]. | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
<div style="background-color:#fffaf0;"> | <div style="background-color:#fffaf0;"> | ||
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | ||
</div> | </div> | ||
<div class="pdbe-citations 3drc" style="background-color:#fffaf0;"></div> | |||
==See Also== | ==See Also== | ||
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__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
[[Category: Bacillus coli migula 1895]] | |||
[[Category: Dihydrofolate reductase]] | [[Category: Dihydrofolate reductase]] | ||
[[Category: Kraut, J]] | [[Category: Kraut, J]] | ||
[[Category: Oatley, S J]] | [[Category: Oatley, S J]] | ||
[[Category: Oxidoreductase]] | [[Category: Oxidoreductase]] | ||
Revision as of 02:22, 5 August 2016
INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESISINVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
Structural highlights
Function[DYR_ECOLI] Key enzyme in folate metabolism. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedWe have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H). The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution. The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain. A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22. Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining. Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range. For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values. While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered. Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme. We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate. Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis.,Warren MS, Brown KA, Farnum MF, Howell EE, Kraut J Biochemistry. 1991 Nov 19;30(46):11092-103. PMID:1932031[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences |
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