Nonadditive Effects of Double Mutations at the Flexible Loops, Glycine-67 and Glycine-121, of Escherichia coli Dihydrofolate Reductase on Its Stability and Function.

• Ohmae Eiji

  Department of Materials Science and Graduate Department of Gene Science, Faculty of Science, Hiroshima University

• Iriyama Koji

  Department of Applied Biological Science, Faculty of Agriculture, Nagoya University

• Ichihara Shigeyuki

  Department of Applied Biological Science, Faculty of Agriculture, Nagoya University

• Gekko Kunihiko

  Department of Materials Science and Graduate Department of Gene Science, Faculty of Science, Hiroshima University

書誌事項

タイトル別名

• Nonadditive Effects of Double Mutations

抄録

The structure, stability, and enzymatic function of dihydrofolate reductase (DHFR) from Escherichia coli are influenced by point mutations at sites 67 and 121 in two flexible loops [Gekko et al. (1994) J. Biochem. 116, 34-41; Ohmae et al. (1996) J. Biochem. 119, 703-710]. In the present study, eight double mutants at sites 67 and 121 (G67V/G121S, G67V/G121A, G67V/G121C, G67V/G121D, G67V/G121V, G67V/G121H, G67V/G121L, and G67V/G121Y) were constructed in order to identify interactions between the two sites of DHFR. The far-ultraviolet circular dichroism spectra of double mutants were clearly different from those of the respective single mutants, with significant changes being observed for three mutants, G67V/G121A, G67V/G121L, and G67V/G121S. The Gibbs free energy change of urea unfolding of double mutants could not be expressed by the sum of those of the respective single mutants except for G67V/G121H. The steady-state kinetic experiments showed that the effect of double mutations manifests itself not in K_m but in k_cat and the transition-state stabilization energy for G67V/G121A, G67V/G121C, and G67V/G121L is not equal to the sum of those for the single mutants. These results indicate that the additivity rule essentially does not hold for these double mutants, and that long-range interactions occur between sites 67 and 121, even though they are separated by 27.7 Å. This is evidence that the flexible loops play important roles in the stability and function of this enzyme through structural perturbations, in which a small alteration in local atomic packing due to amino acid substitution is cooperatively magnified over almost the whole molecule.

収録刊行物

• The Journal of Biochemistry

  The Journal of Biochemistry 123 (1), 33-41, 1998

  社団法人 日本生化学会

キーワード

• dihydrofolate reductase
• double mutants
• flexible loops
• long-range interactions
• stability and function

詳細情報

• CRID: 1390282679907326336
• NII論文ID: 130003533324
• NII論文ID: 10005840430
• NII書誌ID: AA00694073
• DOI: 10.1093/oxfordjournals.jbchem.a021913
• COI: 1:CAS:528:DyaK1cXhsVGhsrw%3D
• ISSN: 17562651
• ISSN: 0021924X
• NDL書誌ID: 4395779
• PubMed: 9504406
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I4395779
• Web Site: https://academic.oup.com/jb/article/123/1/33/830180/Nonadditive-Effects-of-Double-Mutations-at-the
• 本文言語コード: en
• データソース種別:
  • JaLC
  • NDL
  • Crossref
  • PubMed
  • CiNii Articles
• 抄録ライセンスフラグ: 使用不可