Site-Directed Mutagenesis of Histidine Residues in the Ethylene-Forming Enzyme from Pseudomonas syringae

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概要

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• The roles of histidine residues in the catalysis of the transformation of 2-oxoglutarate into ethylene via the ethylene-forming enzyme(EEE)from Pseudomonas syringae were studied using site-directed mutagenesis with substitution of glutamine for ten individual histidine residues. The mutant enzymes, which were expressed in Escherichia coli, were purified to homogeneity, and assayed in vitro for K_m, k_<cat> and thermostability. The relative k_<cat> of two mutated EFEs, H305Q, were 40% and 60%, respectively. However, a mutation at either His-189 or His-233 caused a total loss of activity, implying that these residues play important roles in the binding of iron. The k_<cat> values for other mutant enzymes were 11-to 55-fold less than that for the wild-type enzyme. For six partially inactive mutated EFEs(but not for H305Q or H335Q), the first order rate constants for heat inactivation at 30℃ were 11- to 24-fold higher than for the wild-type enzyme. It is noteworthy that the value of the first order rate constant for heat inactivation of H268Q was identical to that of H335Q. The substitution of H268 resulted in a drastic decrease of the k_<cat> value(relative k_<cat> was 1.8%). This suggests that the substitution at His-268 may cause the disruption of the active site of the EFE. Heat inactivation studies with the purified mutant enzymes revealed that some mutant enzymes, such as H168Q and H116Q, were more thermolabile than the wild-type enzyme.

• 社団法人日本生物工学会の論文
• 1998-03-25

著者

• NAGAHAMA KAZUHIRO

  Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo Universi

• MATSUOKA MASAYOSHI

  Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo Universi

• OGAWA TAKAHIRA

  Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo Universi

• 中村 薫

  京大 化研

• Ogawa T

  Shinshu Univ. Nagano Jpn

• Fukuda H

  Kobe Univ. Kobe

• Fukuda Hideki

  Department Of Molecular Science And Material Engineering Graduate School Of Science And Technology K

• Ogawa T

  Kyoto Univ. Kyoto Jpn

• Ogawa Takahiro

  Department Of Biological Science And Technology Science University Of Tokyo

• Ogawa T

  Department Of Applied Microbial Technology Faculty Of Biotechnology And Life Science Sojo University

• Matsuoka M

  Department Of Applied Microbial Technology Faculty Of Biotechnology And Life Science Sojo University

• Matsuoka Masayoshi

  Dept. Applied Microb. Technol. Sojo Univ.

• Nagahama Kazuhiro

  Department Of Applied Microbial Technology Faculty Of Biotechnology And Life Science Sojo University

• FUKUDA HIDEO

  Department of Applied Microbial Technology, Kumamoto Institute of Technology

• YOSHINO KUNIAKI

  Department of Applied Microbial Technology

• TANASE SUMIO

  Department of Biochemistry, Kumamoto University School of Medicine

• Fukuda H

  Kobe Univ. Kobe Jpn

• Ogawa Takahira

  Department Of Applied Microbial Technology Faculty Of Biotechnology And Life Science Sojo University

• Ogawa Takahira

  Dept. Appl. Microbial Technology Kumamoto Institute Of Technology

• Nagahama Kazuhiro

  Department Of Applied Microbial Technology The Kumanoto Institute Of Technology

• Matsuoka Masayoshi

  Dept. Appl. Microbial Technology Kumamoto Institute Of Technology

• Matsuoka Masayoshi

  Department Of Applied Microbial Technology Faculty Of Biotechnology And Life Science Sojo University

• Fukuda Hideo

  Department Of Applied Microbial Technology The Kumamoto Institute Of Technolgy

• Fukuda Hideo

  Dept. Appl. Microbial Technology Kumamoto Institute Of Technology

• Takegawa Sumio

  Department Of Microbiology Kinki University School Of Medicine

• Ogawa Tadashi

  Graduate School Of Bioagricultural Sciences Nagoya University

• Tanase Sumio

  Department Of Biochemistry Kumamoto University School Of Medicine

• Nakamura Kahoru

  Department Of Nutrition Tokyo University Of Agriculture

• Fukuda Hideo

  Department Of Applied Microbial Technology Kumamoto Institute Of Technology

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