2drc: Difference between revisions

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~~[[Image:2drc.gif|left|200px]]~~

~~{{Structure~~

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

~~|PDB~~= 2drc |~~SIZE~~=~~350|CAPTION~~= <~~scene name~~='~~initialview01~~'>2drc</~~scene~~>, ~~resolution~~ 1.9&~~Aring~~;

|~~SITE~~=

== Structural highlights ==

~~|LIGAND~~= <scene name='pdbligand=CL:~~CHLORIDE~~+ION'>CL</scene>, <scene name='pdbligand=CA:~~CALCIUM~~+ION'>CA</scene> ~~and~~ <scene name='pdbligand=MTX:METHOTREXATE'>MTX</scene>

<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>

~~|ACTIVITY~~= [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]

</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.9Å</td></tr>

|~~GENE~~=

<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>

}}

<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>

</table>

== Function ==

[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.

== Evolutionary Conservation ==

[[Image:Consurf_key_small.gif|200px|right]]

Check<jmol>

<scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/dr/2drc_consurf.spt"</scriptWhenChecked>

<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>

<text>to colour the structure by Evolutionary Conservation</text>

</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/main_output.php?pdb_ID=2drc ConSurf].

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

==See Also==

*[[Dihydrofolate reductase 3D structures|Dihydrofolate reductase 3D structures]]

__TOC__

~~==Overview==~~

</StructureSection>

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.

==~~About this Structure~~==

~~2DRC is a~~ [[~~Single protein]] structure of sequence from [http://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~~].

~~==Reference==~~

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://www.ncbi.nlm.nih.gov/pubmed/~~1932031 1932031]~~

~~[[Category: Dihydrofolate reductase]]~~

[[Category: Escherichia coli]]

[[Category: ~~Single protein~~]]

[[Category: Large Structures]]

[[Category: Brown~~, K A.~~]]

[[Category: Brown KA]]

[[Category: Kraut, J.]]

[[Category: Kraut J]]

~~[[Category: CA]]~~

~~[[Category: CL]]~~

~~[[Category: MTX]]~~

~~[[Category: oxidoreductase]]~~

~~''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 16:30:32 2008''~~

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@@ Line 1: / Line 1: @@
-[[Image:2drc.gif|left|200px]]
- {{Structure
+==INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS==
-|PDB= 2drc |SIZE=350|CAPTION= <scene name='initialview01'>2drc</scene>, resolution 1.9&Aring;
+<StructureSection load='2drc' size='340' side='right'caption='[[2drc]], [[Resolution|resolution]] 1.90&Aring;' scene=''>
-|SITE=
+== Structural highlights ==
-|LIGAND= <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=CA:CALCIUM+ION'>CA</scene> and <scene name='pdbligand=MTX:METHOTREXATE'>MTX</scene>
+<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>
-|ACTIVITY= [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]
+</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>
-|GENE=
+<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>
-}}
+<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>
+</table>
+== Function ==
+[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.
+== Evolutionary Conservation ==
+[[Image:Consurf_key_small.gif|200px|right]]
+Check<jmol>
+  <jmolCheckbox>
+    <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/dr/2drc_consurf.spt"</scriptWhenChecked>
+    <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
+    <text>to colour the structure by Evolutionary Conservation</text>
+  </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/main_output.php?pdb_ID=2drc ConSurf].
+<div style="clear:both"></div>
-'''INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS'''
+==See Also==
+*[[Dihydrofolate reductase 3D structures|Dihydrofolate reductase 3D structures]]
+__TOC__
-==Overview==
+</StructureSection>
-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.
-==About this Structure==
-DRC is a [[Single protein]] structure of sequence from [http://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].
-==Reference==
-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://www.ncbi.nlm.nih.gov/pubmed/1932031 1932031]
-[[Category: Dihydrofolate reductase]]
 [[Category: Escherichia coli]]
-[[Category: Single protein]]
+[[Category: Large Structures]]
-[[Category: Brown, K A.]]
+[[Category: Brown KA]]
-[[Category: Kraut, J.]]
+[[Category: Kraut J]]
-[[Category: CA]]
-[[Category: CL]]
-[[Category: MTX]]
-[[Category: oxidoreductase]]
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 16:30:32 2008''