Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis.
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.
Warren
M S
MS
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Brown
K A
KA
Farnum
M F
MF
Howell
E E
EE
Kraut
J
J
D003067:Coenzymes / Q000737:chemistry; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008727:Methotrexate / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D003067:Coenzymes; D006639:Histidine; D009249:NADP; D014364:Tryptophan; D003903:Deuterium; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate