Effects of point mutations at the flexible loop alanine-145 of Escherichia coli dihydrofolate reductase on its stability and function.
To elucidate the role of a flexible loop (residues 142-149) in the stability and function of Escherichia coli dihydrofolate reductase, alanine-145 in this loop was substituted by site-directed mutagenesis with ten amino acids (Glu, Phe, Gly, His, Ile, Leu, Arg, Ser, Thr, and Val). The amount of three mutant proteins (A145E, A145I, and A145L) in cells was too small to allow the measurement of circular dichroism (CD) spectra and urea unfolding. The CD spectra of other seven mutants were identical with those of the wild-type DHFR, indicating that the native conformation of DHFR was not affected by the mutations. The free energy change of unfolding by urea decreased with an increase in the hydrophobicity of amino acid residues introduced, A145T>A145R>A145G>=A145S>=A145H>A145V++ +>wild-type>=A145F. The steady-state kinetic parameters for the enzyme reaction, Km and ksub, were only slightly influenced by the mutations. These results suggest that site 145 in the flexible loop plays an important role in the stability but has little or no effect on the native structure and function of this enzyme. The characteristics of the mutations are discussed in comparison with those of mutations at site 67 [Ohmae et al. (1996) J. Biochem. 119, 703-710] and at site 121 [Gekko et al. (1994) J. Biochem. 116, 34-41] in two other flexible loops.
Ohmae
E
E
Department of Materials Science and Graduate Department of Gene Science, Faculty of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
Ishimura
K
K
Iwakura
M
M
Gekko
K
K
D000409:Alanine / Q000737:chemistry*; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
C010920:dihydrofolate; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D000409:Alanine