title: Photoreactive nitrile hydratase: the photoreaction site is located on the alpha subunit.
abstract: 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 6 M urea, and two kinds of subunits (alpha and beta) were separated and purified by anion-exchange chromatography. In a manner similar to the native NHase, the isolated alpha subunit showed two absorption peaks at 280 and 370 nm, which were diminished by light irradiation. However, irradiation failed to elicit the appearance of absorption peaks at around 400 nm and at 710 nm, which were characteristic of the activated enzyme. The beta 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 and after light irradiation, but the inactive NHase was reconstituted by incubating the two subunits together in the dark at 4 degrees C for 1 h. Light irradiation of the beta subunit did not affect subsequent complex formation or NHase activity. However, the irradiated alpha subunit could not assemble with the beta subunit, and no activity was recovered. These results demonstrate that the chromophore(s) responsible for the photoactivation of NHase are entirely located on the alpha subunit, and imply that light irradiation induces conformational change of the alpha subunit.
authors.affiliation: Chemical Engineering Laboratory, Institute of Physical and Chemical Research (RIKEN), Saitama.
authors.forename: M
authors.initials: M
authors.lastname: Tsujimura
authors.forename: M
authors.initials: M
authors.lastname: Odaka
authors.forename: S
authors.initials: S
authors.lastname: Nagashima
authors.forename: M
authors.initials: M
authors.lastname: Yohda
authors.forename: I
authors.initials: I
authors.lastname: Endo
mesh_terms: D001665:Binding Sites; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006836:Hydro-Lyases / Q000737:chemistry* / Q000528:radiation effects; D010777:Photochemistry; D011487:Protein Conformation; D012240:Rhodococcus / Q000201:enzymology; D013054:Spectrophotometry, Atomic
chemical_list: D006836:Hydro-Lyases; C048831:nitrile hydratase