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 sta bility and function of Escherichia coli dihydrofolate reductase, alani ne-145 in this loop was substituted by site-directed mutagenesis with ten amino acids (Glu, Phe, Gly, His, lie, Leu, Arg, Ser, Thr, and Val) , The amount of three mutant proteins (A145E, A145I, and A145L) in cel ls was too small to allow the measurement of circular dichroism (CD) s pectra 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 ener gy change of unfolding by urea decreased with an increase in the hydro phobicity of amino acid residues introduced, A145T > A145R > A145G gre ater than or equal to A145S greater than or equal to A145H > A145V > w ild-type greater than or equal to A145F. The steady-state kinetic para meters for the enzyme reaction, K-m and k(cat), were only slightly inf luenced by the mutations. These results suggest that site 145 in the f lexible loop plays an important role in the stability but has little o r no effect on the native structure and function of this enzyme. The c haracteristics 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 o ther flexible loops.