Photoreactive Nitrile Hydratase: The Photoreaction Site Is Located on the α Subunit
Photoreactive Nitrile Hydratase: The Photoreaction Site Is Located on the α Subunit
复制标题
光反应性腈水合酶：光反应位点位于α亚基上
DOI：
--
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
I. Endo
中科院分区：
文献类型：
--
作者：
M. Tsujimura;M. Odaka;S. Nagashima;M. Yohda;I. Endo
红球菌N-771腈水合酶（Nitrile hydratase，NHase）以活性和非活性两种形式存在。无活性的腈水合酶在光照下立即被激活，并转变为活性形式。为了表征光反应中心，用6 M尿素变性失活的腈水合酶，并通过阴离子交换色谱分离和纯化两种亚基（α和β）。以类似于天然腈水合酶的方式，分离的α亚基在280和370 nm处显示出两个吸收峰，其通过光照射而减弱。然而，照射未能引起在400 nm和710 nm附近的吸收峰的出现，这是活化的酶的特征。β亚基似乎不具有任何光反应性发色团，因为它的吸收光谱不被光照射改变。在光照射前后，两个亚基都没有表现出腈水合酶活性，但是通过将两个亚基在4 ℃下在黑暗中一起孵育1小时，可以重建无活性的腈水合酶。光照射的β亚基不影响随后的复合物的形成或腈水合酶活性。然而，辐照的α亚基不能与β亚基组装，并且没有恢复活性。这些结果表明，负责光活化的NHase的发色团完全位于α亚基上，并且暗示光照射诱导α亚基的构象变化。
: 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.