LONG-RANGE STRUCTURAL EFFECTS IN A 2ND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE-REDUCTASE

X-ray crystal structures have been determined for a second-site revert ant (Asp-27 --> Ser, Phe-137 --> Ser; D27S/F137S) and both component s ingle-site mutants of Escherichia coli dihydrofolate reductase. The pr imary D27S mutation, located in the substrate binding pocket, greatly reduces catalytic activity as compared to the wild-type enzyme. The ad ditional F137S mutation, which partially restores catalytic activity, is located on the surface of the molecule, well outside of the catalyt ic center and almost-equal-to 15 angstrom from residue 27. Comparison of kinetic data for the single-site F137S mutant, specifically constru cted as a control, and for the double-mutant enzymes indicates that th e 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 polype ptide backbone. To investigate the mechanism by which the F137S substi tution 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 i nterpretation for the F137S single-site mutant. We find that while eit her single mutant shows little more than a simple side-chain substitut ion, the double mutant undergoes an extended structural perturbation, which is propagated between these two widely separated sites via the h elix alphaB.