3drc
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INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
OverviewOverview
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 StructureAbout this Structure
3DRC is a Single protein structure of sequence from Escherichia coli with CL, CA and MTX as ligands. Active as Dihydrofolate reductase, with EC number 1.5.1.3 Full crystallographic information is available from OCA.
ReferenceReference
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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