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J. Biochem, 1996, Vol. 119, No. 4 703-710
© 1996 Japanese Biochemical Society

research-article

Effects of Point Mutations at the Flexible Loop Glycine-67 of Escherichia coli; Dihydrofolate Reductase on its Stability and Function

Eiji Ohmae, Koji Iriyama, Shigeyuki Ichihara and Kunihiko Gekko1

Department of Applied Biological Science, Faculty of Agriculture, Nagoya University Chikusa-ku, Nagoya, Aichi 464-01

1To whom correspondence should be addressed at the present address: Department of Materials Science and Graduate Department of Gene Science, Faculty of Science, Hiroshima University, Kagami-yama, Higashi-Hiroshima 739.

To elucidate the role of a flexible loop (residues 64-72) in the stability and function ofEscherichia coli dihydrofolate reductase, glycine-67 in this loop was substituted by site-directed mutagenesis with seven amino acids (Ala, Cys, Asp, Leu, Ser, Thr, and Val). The circular dichroism spectra suggested that the conformation of the native structure was affected by the mutations in both the presence and absence of NADPH. The free energy change of unfolding by urea decreased in the order of G67A>G67S≥wild-type greater double equalsG67D> G67T≥G67C>G67L>G67V. The steady-state kinetic parameters for the enzyme reaction, Km; and kcat;, were only slightly influenced, but the rate of the hydride transfer reaction was significantly changed by the mutations, as revealed by the deuterium isotope effect on the enzyme activity. These results suggest that site 67 in the flexible loop, being very far from the active site, plays an important role in the stability and function of this enzyme. The characteristics of the mutations were discussed in terms of the modified flexibility of the native structure, compared with the results of mutations at site 121 in another flexible loop [Gekko et al. (1994)J. Biochem. 116, 34–41].


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