Photoreactive Nitrile Hydratase: The Photoreaction Site Is Located on the .ALPHA. Subunit.

概要

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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.
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