In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii
In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii
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维氏固氮菌 [NiFe] 氢化酶的体内和体外镍依赖性加工
DOI：
10.1128/jb.176.2.291-295.1994
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发表时间：
1994
影响因子：
3.2
通讯作者：
Robert L. Robson
中科院分区：
文献类型：
--
作者：
A. Menon;Robert L. Robson
棕色固氮菌中的氢氧化是由膜结合的α-β二聚体[NiFe]氢酶催化的。该酶的成熟包括切割β亚基中可能的N-末端信号序列，并从α亚基的C末端去除15个氨基酸。仅限于镍的细胞表现出较低的氢酶活性，并含有明显较大形式的阿尔法亚单位。在这类细胞中加入镍使氢酶活性在2小时内增加了5倍。第一小时的增加不需要转录和翻译，并且与从纯化的酶中提取的大形式的α亚基(前α)到类似于α亚基的小形式(α)的加工有关。在体内，前α蛋白表现为可溶的，而大部分α蛋白是膜结合的。在镍限制细胞的膜耗尽提取液中，体外复制了从前阿尔法到阿尔法的过程。加工特别需要添加Ni2+，而Co2+、Cu2+、Ca2+、Fe2+、Mn2+和Zn2+无效。然而，锌离子、Co2+和Cu2+抑制镍依赖的加工。镁-三磷酸腺苷和镁-GTP促进了处理，而厌氧条件和/或添加二硫苏糖醇和二亚硫酸钠是不必要的。蛋白水解酶抑制剂苯甲基磺酰氟、E64和胃抑素不抑制这一过程。
H2 oxidation in Azotobacter vinelandii is catalyzed by a membrane-bound, alpha beta dimeric [NiFe] hydrogenase. Maturation of the enzyme involves cleavage of a putative N-terminal signal sequence in the beta subunit and removal of 15 amino acids from the C terminus of the alpha subunit. Cells limited for nickel exhibited low hydrogenase activities and contained an apparently large form of the alpha subunit. Addition of nickel to such cells increased hydrogenase activities fivefold over 2 h. The increase in the first hour did not require transcription and translation and correlated with processing of the large form of the alpha subunit (pre-alpha) to the small form (alpha) resembling the alpha subunit from the purified enzyme. In vivo, pre-alpha appeared soluble whereas the majority of alpha was membrane bound. Processing of pre-alpha to alpha was reproduced in vitro in membrane-depleted extracts of nickel-limited cells. Processing specifically required the addition of Ni2+, whereas Co2+, Cu2+, Ca2+, Fe2+, Mn2+, and Zn2+ were ineffective. However, Zn2+, Co2+, and Cu2+ inhibited nickel-dependent processing. Mg-ATP and Mg-GTP stimulated processing, whereas anaerobic conditions and/or the addition of dithiothreitol and sodium dithionite was unnecessary. Processing was not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, E64, and pepstatin.
影响因子：
11.3
作者：
A. Przybyla;Jeffery Robbins;N. Menon;H. D. Peck
通讯作者：
A. Przybyla;Jeffery Robbins;N. Menon;H. D. Peck