EFFECTS OF POINT MUTATIONS AT THE FLEXIBLE LOOP GLYCINE-67 OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE ON ITS STABILITY AND FUNCTION

To elucidate the role of a flexible loop (residues 64-72) in the stabi lity and function of Escherichia call dihydrofolate reductase, glycine -67 in this loop was substituted by site-directed mutagenesis with sev en amino acids (Ala, Cys, Asp, Leu, Ser, Thr, and Val). The circular d ichroism spectra suggested that the conformation of the native structu re was affected by the mutations in both the presence and absence of N ADPH. The free energy change of unfolding by urea decreased in the ord er of G67A > G67S greater than or equal to wild-type greater than or e qual to G67D > G67T > G67C greater than or equal to G67L > G67V. The s teady-state kinetic parameters for the enzyme reaction, K-m and k(cat) , were only slightly influenced, but the rate of the hydride transfer reaction was significantly changed by the mutations, as revealed by th e deuterium isotope effect on the enzyme activity. These results sugge st that site 67 in the flexible loop, being very far from the active s ite, plays an important role in the stability and function of this enz yme. The characteristics of the mutations were discussed in terms of t he modified flexibility of the native structure, compared with the res ults of mutations at site 121 in another flexible loop [Gekko et al, ( 1994) J. Biochem, 116, 34-41].