Random Mutagenesis of Glutamine Synthetase from Escherichia coli; Correlation between Structure, Activity, and Fitness

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概要

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• Glutamine synthetase is a key enzyme in nitrogen metabolism and has been improved during the course of evolution. The present level of its activity was evaluated by comparing with the activities of mutant enzymes prepared by rendom mutagenesis. With respect to the transferase activity, only 3% of a total of 73688 mutants had markadly higher activity, to the extent of 2.7 times higher than that of the wild-type. These results show that the level of the activity of the wild-type enzyme is high and well optimized, through it is not the highest. Purified preparations of the wild-type enzyme (GLS-W) and two selected mutant enzymes (GLS-H and GLS-L) showed specific transferase activities (units/mg protein) of 84.5,184 and 7.1,respectively. For synthetase activity, the k_<cat> values (s^<-1>) were : GLS-W, 25.8; GLS-H, 300; GLS, 23.2; the K_m values (mM) for L-gutamate were : GLS-W, 2.6; GLS-H, 10.7; GLS-L, 14.6. The numbers of adenylyl groups bound to the enzyme were estimated to be : GLS-W, 11; GLS-H, 0; GLS-L, 7. GLS-H had a mutation of Tyr-397 to His, while GLS-L had two amino acid chnages from Ala-35 to Val and Pro-94 to Leu. These residues are located on the surface of the dodecamer molecule. The distances between the active site Mn^<2+> ion and the α-carbon atoms of the mutated residues are longer than 20 Å. Tyr-397 is known to be the target site for the regulation of activity by adenylylation. Finally, the effect of the difference in the activity of glutamine synthetase on the specific growth rate of Escherichia coli was examined. E. coli expressing GLS-W showed higher specific rate than that expressing GLS-H or GLS-L.

• 社団法人日本生物工学会の論文
• 1994-03-25

著者

• Yomo Tetsuya

  Department of Bioinformatics Engineering, Graduate School of Information Science and Technology, Osa

• Yomo T

  Osaka Univ. Osaka Jpn

• Yomo Tetsuya

  Department Of Bioinformatic Engineering Graduate School Of Information Science And Technology Osaka

• Urabe I

  Osaka Univ. Osaka Jpn

• Urabe Itaru

  Department Of Biotechnology Graduate School Of Engineering Osaka University

• Urabe I

  Graduate School Of Engineering Osaka University

• Urabe Itaru

  Department Of Biotechnology Faculty Of Engineering Osaka University

• Yamamoto K

  Graduate School Of Biostudies Kyoto University

• Yomo Tetsuya

  Department Of Bioinformatic Engineering Graduate School Of Information Science And Technology Osaka

• Urabe Itaru

  Dept. Biotech. Grad. Sch. Eng. Osaka Univ.

• Yamamoto Keizo

  Department of Cardiovascular Medicine, Cardiovascular Center Sakakibara Hospital

• Urabe Itaru

  Department Of Biotechnology Osaka University

• ZHONG-XU WEI

  Department of Biotechnology, Faculty of Engineering, Osaka University

• FUKIHARA JUN

  Department of Biotechnology, Faculty of Engineering, Osaka University

• Zhong-xu Wei

  Department Of Biotechnology Faculty Of Engineering Osaka University

• Yamamoto Keizo

  Department Of Anatomy Nara Medical University:department Of Anatomy School Of Medicine Tokushima Uni

• Fukihara Jun

  Department Of Biotechnology Faculty Of Engineering Osaka University

• Urabe Itaru

  Graduate School Of Engineering Osaka University

• Yomo Tetsuya

  Erato Jst:graduate School Of Information Science And Technology Osaka University:graduate School Of

• Yamamoto K

  Department Of Chemistry Nara Medical University

• Yomo Tetsuya

  Department Of Bioinformatics Engineering Graduate School Of Information Science And Technology Osaka

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