Photoreactive Nitrile Hydratase: The Photoreaction Site Is Located on the Subunit
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- Tsujimura Masanari
- Graduate School of Science and Engineering, Saitama University
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- Odaka Masafumi
- Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN).
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- Nagashima Shigehiro
- Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN).
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- Yohda Masafumi
- Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN).
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- Endo Isao
- Graduate School of Science and Engineering, Saitama University Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN).
書誌事項
- タイトル別名
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- Photoreactive Nitrile Hydratase: The Photoreaction Site Is Located on the .ALPHA. Subunit.
- Photoreactive nitrile hydratase: the photoreaction site is located on the α subunit
抄録
Nitrile hydratase (NHase) from Rhodococcus sp. N-771 exists in active and inactive forms. The inactive NHase is immediately activated by light irradiation and changes to the active form. To characterize the photoreactive center, the inactive NHase was denatured by 6M urea, and two kinds of subunits (α and β) were separated and purified by anion-exchange chromatography. In a manner similar to the native NHase, the isolated α subunit showed two absorption peaks at 280 and 370nm, which were diminished by light irradiation. However, irradiation failed to elicit the appearance of absorption peaks at around 400nm and at 710nm, which were characteristic of the activated enzyme. The β subunit seemed not to possess any photoreactive chromophore because its absorption spectrum was not altered by light irradiation. Neither of the subunits showed NHase activity before or after light irradiation, but the inactive NHase was reconstituted by incubating the two subunits together in the dark at 4°C for 1h. Light irradiation of the β subunit did not affect subsequent complex formation or NHase activity. However, the irradiated α subunit could not assemble with the β subunit, and no activity was recovered. These results demonstrate that the chromophore (s) responsible for the photoactivation of NHase are entirely located on the α subunit, and imply that light irradiation induces conformational change of the α subunit.
収録刊行物
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- The Journal of Biochemistry
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The Journal of Biochemistry 119 (3), 407-413, 1996
社団法人 日本生化学会
