1DHI

LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE

Summary for 1DHI

• Entry DOI: 10.2210/pdb1dhi/pdb
• Descriptor: DIHYDROFOLATE REDUCTASE, CHLORIDE ION, METHOTREXATE, ... (5 entities in total)
• Functional Keywords: oxidoreductase
• Biological source: Escherichia coli
• Total number of polymer chains: 2
• Total formula weight: 37004.49

Authors

Oatley, S.J.,Villafranca, J.E.,Brown, K.A.,Kraut, J. (deposition date: 1993-10-29, release date: 1994-01-31, Last modification date: 2024-02-07)

Primary citation

Brown, K.A.,Howell, E.E.,Kraut, J.
Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.
Proc.Natl.Acad.Sci.USA, 90:11753-11756, 1993

PubMed 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.

PubMed: 8265622
DOI: 10.1073/pnas.90.24.11753

Experimental method

X-RAY DIFFRACTION (1.9 Å)