Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.
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.
Brown
K A
KA
Department of Chemistry, University of California at San Diego, La Jolla 92093.
Howell
E E
EE
Kraut
J
J
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D001224:Aspartic Acid; D012694:Serine; D010649:Phenylalanine; D013762:Tetrahydrofolate Dehydrogenase