2drc
From Proteopedia
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|PDB= 2drc |SIZE=350|CAPTION= <scene name='initialview01'>2drc</scene>, resolution 1.9Å | |PDB= 2drc |SIZE=350|CAPTION= <scene name='initialview01'>2drc</scene>, resolution 1.9Å | ||
|SITE= | |SITE= | ||
| - | |LIGAND= <scene name='pdbligand= | + | |LIGAND= <scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=MTX:METHOTREXATE'>MTX</scene> |
| - | |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] | + | |ACTIVITY= <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> |
|GENE= | |GENE= | ||
| + | |DOMAIN= | ||
| + | |RELATEDENTRY= | ||
| + | |RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=2drc FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2drc OCA], [http://www.ebi.ac.uk/pdbsum/2drc PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=2drc RCSB]</span> | ||
}} | }} | ||
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[[Category: Brown, K A.]] | [[Category: Brown, K A.]] | ||
[[Category: Kraut, J.]] | [[Category: Kraut, J.]] | ||
| - | [[Category: CA]] | ||
| - | [[Category: CL]] | ||
| - | [[Category: MTX]] | ||
[[Category: oxidoreductase]] | [[Category: oxidoreductase]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Mar 31 02:38:24 2008'' |
Revision as of 23:38, 30 March 2008
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| , resolution 1.9Å | |||||||
|---|---|---|---|---|---|---|---|
| Ligands: | , , | ||||||
| Activity: | Dihydrofolate reductase, with EC number 1.5.1.3 | ||||||
| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||
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. 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
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