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2drc

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(New page: 200px<br /><applet load="2drc" size="450" color="white" frame="true" align="right" spinBox="true" caption="2drc, resolution 1.9&Aring;" /> '''INVESTIGATION OF THE ...)
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[[Image:2drc.gif|left|200px]]<br /><applet load="2drc" size="450" color="white" frame="true" align="right" spinBox="true"
 
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caption="2drc, resolution 1.9&Aring;" />
 
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'''INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS'''<br />
 
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==Overview==
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==INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS==
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We 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.
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<StructureSection load='2drc' size='340' side='right'caption='[[2drc]], [[Resolution|resolution]] 1.90&Aring;' scene=''>
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== Structural highlights ==
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<table><tr><td colspan='2'>[[2drc]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2DRC OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2DRC FirstGlance]. <br>
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</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.9&#8491;</td></tr>
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<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><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>
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<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=2drc FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2drc OCA], [https://pdbe.org/2drc PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2drc RCSB], [https://www.ebi.ac.uk/pdbsum/2drc PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2drc ProSAT]</span></td></tr>
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</table>
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== Function ==
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[https://www.uniprot.org/uniprot/DYR_ECOLI DYR_ECOLI] Key enzyme in folate metabolism. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis.
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== Evolutionary Conservation ==
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[[Image:Consurf_key_small.gif|200px|right]]
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Check<jmol>
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<jmolCheckbox>
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<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/dr/2drc_consurf.spt"</scriptWhenChecked>
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<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
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<text>to colour the structure by Evolutionary Conservation</text>
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</jmolCheckbox>
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</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=2drc ConSurf].
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<div style="clear:both"></div>
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==About this Structure==
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==See Also==
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2DRC is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with CL, CA and MTX as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [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] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2DRC OCA].
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*[[Dihydrofolate reductase 3D structures|Dihydrofolate reductase 3D structures]]
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__TOC__
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==Reference==
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</StructureSection>
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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:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=1932031 1932031]
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[[Category: Dihydrofolate reductase]]
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[[Category: Escherichia coli]]
[[Category: Escherichia coli]]
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[[Category: Single protein]]
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[[Category: Large Structures]]
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[[Category: Brown, K.A.]]
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[[Category: Brown KA]]
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[[Category: Kraut, J.]]
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[[Category: Kraut J]]
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[[Category: CA]]
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[[Category: CL]]
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[[Category: MTX]]
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[[Category: oxidoreductase]]
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''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 09:44:33 2007''
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Current revision

INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS

PDB ID 2drc

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