A second-site mutation at phenylalanine-137 that increases catalytic efficiency in the mutant aspartate-27----serine Escherichia coli dihydrofolate reductase.
The adaptability of Escherichia coli dihydrofolate reductase (DHFR) is being explored by identifying second-site mutations that can partially suppress the deleterious effect associated with removal of the active-site proton donor aspartic acid-27. The Asp27----serine mutant DHFR (D27S) was previously characterized and the catalytic activity found to be greatly decreased at pH 7.0 [Howell et al. (1986) Science 231, 1123-1128]. Using resistance to trimethoprim (a DHFR inhibitor) in a genetic selection procedure, we have isolated a double-mutant DHFR gene containing Asp27----Ser and Phe137----Ser mutations (D27S+F137S). The presence of the F137S mutation increases kcat approximately 3-fold and decreases Km(DHF) approximately 2-fold over D27S DHFR values. The overall effect on kcat/Km(DHF) is a 7-fold increase. The D27S+F137S double-mutant DHFR is still 500-fold less active than wild-type DHFR at pH 7. Surprisingly, Phe137 is approximately 15 A from residue 27 in the active site and is part of a beta-bulge. We propose the F137S mutation likely causes its catalytic effect by slightly altering the conformation of D27S DHFR. This supposition is supported by the observation that the F137S mutation does not have the same kinetic effect when introduced into the wild-type and D27S DHFRs, by the altered distribution of two conformers of free enzyme [see Dunn et al. (1990)] and by a preliminary difference Fourier map comparing the D27S and D27S+F137S DHFR crystal structures.
Howell
E E
EE
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Booth
C
C
Farnum
M
M
Kraut
J
J
Warren
M S
MS
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005813:Genes, Synthetic; D007700:Kinetics; D008958:Models, Molecular; D010649:Phenylalanine; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014295:Trimethoprim / Q000378:metabolism / Q000494:pharmacology
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010649:Phenylalanine; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase