caa a22 4500 397557485 CHVBK 20180308164807.0 cr unu---uuuuu 161202e199604 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021299 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021299 Effects of Point Mutations at the Flexible Loop Glycine-67 of Escherichia coli; Dihydrofolate Reductase on its Stability and Function [Elektronische Daten] [Eiji Ohmae, Koji Iriyama, Shigeyuki Ichihara, Kunihiko Gekko] 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 ≧G67D> 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]. © by the Journal of Biochemistry Regular Papers nationallicence dihydrofolate reductase nationallicence enzyme function nationallicence point mutation nationallicence role of flexible loop nationallicence structural stability nationallicence Ohmae Eiji Department of Applied Biological Science, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-01 aut Iriyama Koji Department of Applied Biological Science, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-01 aut Ichihara Shigeyuki Department of Applied Biological Science, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-01 aut Gekko Kunihiko Department of Applied Biological Science, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-01 aut The Journal of Biochemistry Oxford University Press 119/4(1996-04), 703-710 0021-924X 119:4<703 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021299 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021299 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ohmae Eiji Department of Applied Biological Science, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-01 aut NATIONALLICENCE 700 1- Iriyama Koji Department of Applied Biological Science, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-01 aut NATIONALLICENCE 700 1- Ichihara Shigeyuki Department of Applied Biological Science, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-01 aut NATIONALLICENCE 700 1- Gekko Kunihiko Department of Applied Biological Science, Faculty of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-01 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/4(1996-04), 703-710 0021-924X 119:4<703 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence SWISSBIB 397557485 BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford