2drc
From Proteopedia
(New page: 200px<br /><applet load="2drc" size="450" color="white" frame="true" align="right" spinBox="true" caption="2drc, resolution 1.9Å" /> '''INVESTIGATION OF THE ...) |
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| - | [[Image:2drc.gif|left|200px]]<br /><applet load="2drc" size=" | + | [[Image:2drc.gif|left|200px]]<br /><applet load="2drc" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="2drc, resolution 1.9Å" /> | caption="2drc, resolution 1.9Å" /> | ||
'''INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS'''<br /> | '''INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS'''<br /> | ||
==Overview== | ==Overview== | ||
| - | We have applied site-directed mutagenesis methods to change the conserved | + | 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== | ==About this Structure== | ||
| - | 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:// | + | 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 <scene name='pdbligand=CL:'>CL</scene>, <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=MTX:'>MTX</scene> 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://oca.weizmann.ac.il/oca-bin/ocashort?id=2DRC OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Brown, K | + | [[Category: Brown, K A.]] |
[[Category: Kraut, J.]] | [[Category: Kraut, J.]] | ||
[[Category: CA]] | [[Category: CA]] | ||
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[[Category: oxidoreductase]] | [[Category: oxidoreductase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:01:46 2008'' |
Revision as of 15:01, 21 February 2008
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INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
Overview
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 Escherichia coli with , and as ligands. Active as Dihydrofolate reductase, with EC number 1.5.1.3 Full crystallographic information is available from 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:1932031
Page seeded by OCA on Thu Feb 21 17:01:46 2008
Categories: Dihydrofolate reductase | Escherichia coli | Single protein | Brown, K A. | Kraut, J. | CA | CL | MTX | Oxidoreductase
