Essential tyrosine residues in 3-ketosteroid-Δ1-dehydrogenase from Rhodococcus rhodochrous

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概要

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• Tetranitromethane treatment of 3-ketosteroid-Δ1-dehydrogenase of Rhodococcus rhodechrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0.014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.

• 日本生化学会 = Japanese Biochemical Societyの論文
• 1999-10-01

著者

• Itagaki E

  Kanazawa Univ. Kanazawa

• Itagaki Eiji

  Departments Of Chemistry Faculty Of Science Kanazawa University

• Itagaki Eiji

  Department Of Chemistry Faculty Of Science Kanazawa University

• Iwami Masafumi

  Departments Of Biology Faculty Of Science Kanazawa University

• MORII Shingo

  Departments of Chemistry and Biology, Fuculty of Science, Kanazawa University

• FUJII Chifumi

  Departments of Chemistry and Biology, Fuculty of Science, Kanazawa University

• KADODE Michiaki

  Departments of Chemistry Faculty of Science, Kanazawa University

• SAWAMOTO Shizue

  Departments of Chemistry Faculty of Science, Kanazawa University

• IWAMI Masafumi

  Biology, Faculty of Science, Kanazawa University

• Fujii Chizuko

  Departments Of Chemistry Faculty Of Science Kanazawa University

• Morii Shingo

  Departments Of Chemistry Faculty Of Science Kanazawa University

• Sawamoto Shizue

  Departments Of Chemistry Faculty Of Science Kanazawa University

• Kadode Michiaki

  Departments Of Chemistry Faculty Of Science Kanazawa University

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