A Single Histidine Is Required for Activity of Cytochrome c Peroxidase from Paracoccus denitrificans(*)
A Single Histidine Is Required for Activity of Cytochrome c Peroxidase from Paracoccus denitrificans(*)
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脱氮副球菌的细胞色素 c 过氧化物酶活性需要单个组氨酸(*)
DOI：
10.1074/jbc.271.19.11126
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
G. Pettigrew
中科院分区：
文献类型：
--
作者：
D. Mcginnity;B. Devreese;Susana Prazeres;J. V. Van Beeumen;I. Moura;J. Moura;G. Pettigrew
用组氨酸特异性试剂焦碳酸二乙酯对副球菌的二血红素细胞色素c过氧化物酶进行修饰。在低过量的试剂下，在氧化酶中修饰1摩尔组氨酸，并且修饰与形成活性状态的能力的丧失相关。随着时间的推移，修改逆转，并恢复形成活性状态的能力。组氨酸修饰的分光光度测量和放射性掺入使用放射性标记试剂之间的协议表明其他氨基酸的修饰很少。然而，组氨酸修饰的逆转观察到的放射性损失不匹配，我们提出了一个缓慢的转移的乙氧基甲酰基到一个身份不明的氨基酸。CN的存在下绑定到酶的活性过氧化物位点导致的基本组氨酸从修改的完全保护。对天然酶进行有限的枯草杆菌蛋白酶处理，然后对C末端片段（残基251-338）进行胰蛋白酶消化，结果表明放射性位于第275位含有单个组氨酸的肽中。我们建议，这个保守的残基，在一个高度保守的区域，是中央的活性混合价态的功能。
The diheme cytochrome c peroxidase from Paracoccus denitrificans was modified with the histidine-specific reagent diethyl pyrocarbonate. At low excess of reagent, 1 mol of histidine was modified in the oxidized enzyme, and modification was associated with loss of the ability to form the active state. With time, the modification reversed, and the ability to form the active state was recovered. The agreement between the spectrophotometric measurement of histidine modification and radioactive incorporation using a radiolabeled reagent indicated little modification of other amino acids. However, the reversal of histidine modification observed spectrophotometrically was not matched by loss of radioactivity, and we propose a slow transfer of the ethoxyformyl group to an unidentified amino acid. The presence of CN bound to the active peroxidatic site of the enzyme led to complete protection of the essential histidine from modification. Limited subtilisin treatment of the native enzyme followed by tryptic digest of the C-terminal fragment (residues 251-338) showed that radioactivity was located in a peptide containing a single histidine at position 275. We propose that this conserved residue, in a highly conserved region, is central to the function of the active mixed-valence state.