3drc: Difference between revisions
No edit summary |
No edit summary |
||
| Line 1: | Line 1: | ||
[[Image:3drc.gif|left|200px]] | [[Image:3drc.gif|left|200px]] | ||
'''INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS''' | {{Structure | ||
|PDB= 3drc |SIZE=350|CAPTION= <scene name='initialview01'>3drc</scene>, resolution 1.9Å | |||
|SITE= | |||
|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> | |||
|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] | |||
|GENE= | |||
}} | |||
'''INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS''' | |||
==Overview== | ==Overview== | ||
| Line 7: | Line 16: | ||
==About this Structure== | ==About this Structure== | ||
3DRC is a [ | 3DRC 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=3DRC OCA]. | ||
==Reference== | ==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:// | 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: Dihydrofolate reductase]] | ||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
| Line 21: | Line 30: | ||
[[Category: oxidoreductase]] | [[Category: oxidoreductase]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 19:04:28 2008'' | ||
Revision as of 17:04, 20 March 2008
| |||||||||||||
| 3drc, resolution 1.9Å | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Ligands: | CL, CA and MTX | ||||||||||||
| Activity: | Dihydrofolate reductase, with EC number 1.5.1.3 | ||||||||||||
| 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
3DRC 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
Page seeded by OCA on Thu Mar 20 19:04:28 2008
