Unknown

Dataset Information

0

Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.


ABSTRACT: X-ray crystal structures have been determined for a second-site revertant (Asp-27-->Ser, Phe-137-->Ser; D27S/F137S) and both component single-site mutants of Escherichia coli dihydrofolate reductase. The primary D27S mutation, located in the substrate binding pocket, greatly reduces catalytic activity as compared to the wild-type enzyme. The additional F137S mutation, which partially restores catalytic activity, is located on the surface of the molecule, well outside of the catalytic center and approximately 15 A from residue 27. Comparison of kinetic data for the single-site F137S mutant, specifically constructed as a control, and for the double-mutant enzymes indicates that the effects of the F137S and D27S mutations on catalysis are nonadditive. This result suggests that the second-site mutation might mediate its effects through a structural perturbation propagated along the polypeptide backbone. To investigate the mechanism by which the F137S substitution elevates the catalytic activity of D27S we have determined the structure of the D27S/F137S double mutant. We also present a rerefined structure for the original D27S mutant and a preliminary structural interpretation for the F137S single-site mutant. We find that while either single mutant shows little more than a simple side-chain substitution, the double mutant undergoes an extended structural perturbation, which is propagated between these two widely separated sites via the helix alpha B.

SUBMITTER: Brown KA 

PROVIDER: S-EPMC48062 | biostudies-other | 1993 Dec

REPOSITORIES: biostudies-other

altmetric image

Publications

Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.

Brown K A KA   Howell E E EE   Kraut J J  

Proceedings of the National Academy of Sciences of the United States of America 19931201 24


X-ray crystal structures have been determined for a second-site revertant (Asp-27-->Ser, Phe-137-->Ser; D27S/F137S) and both component single-site mutants of Escherichia coli dihydrofolate reductase. The primary D27S mutation, located in the substrate binding pocket, greatly reduces catalytic activity as compared to the wild-type enzyme. The additional F137S mutation, which partially restores catalytic activity, is located on the surface of the molecule, well outside of the catalytic center and  ...[more]

Similar Datasets

| S-EPMC2740434 | biostudies-literature
| S-EPMC2765523 | biostudies-literature
| S-EPMC3166973 | biostudies-literature
| S-EPMC5336361 | biostudies-literature
| S-EPMC3773979 | biostudies-literature
| S-EPMC3323781 | biostudies-literature
| S-EPMC556001 | biostudies-literature
| S-EPMC7503464 | biostudies-literature
| S-EPMC7848751 | biostudies-literature
| S-EPMC5773535 | biostudies-literature