caa a22 4500 397558155 CHVBK 20180308164809.0 cr unu---uuuuu 161202e199603 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021256 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021256 Photoreactive Nitrile Hydratase: The Photoreaction Site Is Located on the α Subunit [Elektronische Daten] [Masanari Tsujimura, Masafumi Odaka, Shigehiro Nagashima, Masafumi Yohda, Isao Endo] 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 (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 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 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 1 h. Light irradiation of the β subunit did not affect subsequent complex formation or NHase activity. However, the irradiated a subunit could not assemble with the ff subunit, and no activity was recovered. These results demonstrate that the chromophore(s) responsible for the photoactivation of NHase are entirely located on the a subunit, and imply that light irradiation induces conformational change of the a subunit. © by the Journal of Biochemistry Regular Papers nationallicence nitrile hydratase nationallicence non-heme iron center nationallicence photoreactive enzyme nationallicence reconstitution nationallicence subunit isolation nationallicence Tsujimura Masanari Graduate School of Science and Engineering, Saitama University Urawa, Saitama 338 aut Odaka Masafumi Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako, Saitama 351-01 aut Nagashima Shigehiro Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako, Saitama 351-01 aut Yohda Masafumi Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako, Saitama 351-01 aut Endo Isao Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako, Saitama 351-01 aut The Journal of Biochemistry Oxford University Press 119/3(1996-03), 407-413 0021-924X 119:3<407 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021256 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021256 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Tsujimura Masanari Graduate School of Science and Engineering, Saitama University Urawa, Saitama 338 aut NATIONALLICENCE 700 1- Odaka Masafumi Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako, Saitama 351-01 aut NATIONALLICENCE 700 1- Nagashima Shigehiro Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako, Saitama 351-01 aut NATIONALLICENCE 700 1- Yohda Masafumi Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako, Saitama 351-01 aut NATIONALLICENCE 700 1- Endo Isao Chemical Engineering Laboratory, The Institute of Physical and Chemical Research (RIKEN) 2-1 Hirosawa, Wako, Saitama 351-01 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/3(1996-03), 407-413 0021-924X 119:3<407 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford