title: Effects of point mutations at the flexible loop glycine-67 of Escherichia coli dihydrofolate reductase on its stability and function.
abstract: To elucidate the role of a flexible loop (residues 64-72) in the stability and function of Escherichia 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 confirmation 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 > or = wild-type > or = G67D > G67T > G67C > or = 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.
authors: Department of Applied Biological Science, Faculty of Agriculture, Nagoya University.
authors: E
authors: E
authors: Ohmae
authors: K
authors: K
authors: Iriyama
authors: S
authors: S
authors: Ichihara
authors: K
authors: K
authors: Gekko
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000502:physiology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014508:Urea
chemical_list: D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase; D005998:Glycine