Effects of insertions and deletions in a beta-bulge region of Escherichia coli dihydrofolate reductase.
The role of a beta-bulge in Escherichia coli dihydrofolate reductase (DHFR) has been explored by a series of insertion and deletion mutations. Insertion of a seven amino acid sequence from a structurally equivalent 'beta-blowout' sequence from human DHFR destabilizes E. coli DHFR by 3.6 kcal/mol and decreases catalytic efficiency (kcat/K(m)) 34-fold. Deletion of F137, delta 137, the looped out residue in the bulge, also destabilizes E. coli DHFR by 2.8 kcal/mol but only decreases catalytic efficiency threefold. Concurrent deletion of F137 and mutation of, V136 to proline to try and maintain the strand twist associated with the beta-bulge decreases protein stability by 3.4 kcal/mol and decreases catalytic efficiency 84-fold. These insertion/deletion mutations were also constructed in a D27S DHFR background. The D27S mutation has been described previously and proposed to remove the catalytic acid from the active site. The delta 137 mutation partially suppresses the effect of the D27S mutation as it decreases the K(m) for substrate, dihydrofolate, twofold. Non-additive effects are observed for the insertion/deletion mutations in wild-type versus D27S DHFR backgrounds, consistent with structural changes.
Dion-Schultz
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Department of Biochemistry, University of Tennessee, Knoxville 37996-0840, USA.
Howell
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D002384:Catalysis; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004251:DNA Transposable Elements; D013762:Tetrahydrofolate Dehydrogenase