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| {{STRUCTURE_2drc| PDB=2drc | SCENE= }} | | {{STRUCTURE_2drc| PDB=2drc | SCENE= }} |
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| '''INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS'''
| | ===INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS=== |
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| ==Overview==
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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.
| | The line below this paragraph, {{ABSTRACT_PUBMED_1932031}}, adds the Publication Abstract to the page |
| | (as it appears on PubMed at http://www.pubmed.gov), where 1932031 is the PubMed ID number. |
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| | {{ABSTRACT_PUBMED_1932031}} |
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| ==About this Structure== | | ==About this Structure== |
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| [[Category: Kraut, J.]] | | [[Category: Kraut, J.]] |
| [[Category: Oxidoreductase]] | | [[Category: Oxidoreductase]] |
| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 01:00:16 2008'' | | |
| | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 05:28:03 2008'' |
