A radical on the Met-Tyr-Trp modification required for catalase activity in catalase-peroxidase is established by isotopic labeling and site-directed mutagenesis.
A radical on the Met-Tyr-Trp modification required for catalase activity in catalase-peroxidase is established by isotopic labeling and site-directed mutagenesis.
复制标题
通过同位素标记和定点诱变建立过氧化氢酶-过氧化物酶中过氧化氢酶活性所需的 Met-Tyr-Trp 修饰上的自由基。
DOI：
10.1021/ja103311e
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发表时间：
2010
影响因子：
15
通讯作者：
Magliozzo,RichardS
中科院分区：
文献类型：
--
作者：
Zhao,Xiangbo;Suarez,Javier;Khajo,Abdelahad;Yu,Shengwei;Metlitsky,Leonid;Magliozzo,RichardS
结核分枝杆菌过氧化氢酶-过氧化物酶（KatG）在过氧化氢酶周转过程中产生一个短暂的酪氨酰样自由基，并伴有一个窄的双峰X-带EPR信号。用β-亚甲基-氘代酪氨酸标记KatG会导致双峰塌陷为单峰，而对于3，5-环-氘代酪氨酸标记的酶，EPR信号没有发生变化。除了替换Tyr 229 Phe之外，KatG的所有其他单酪氨酸突变体表现出与野生型酶类似的窄双峰EPR信号和过氧化氢酶活性。这些发现证实了该催化活性基团与Tyr 229相关，其3′和5′质子由于与含有远端侧Met 255-Tyr 229-Trp 107加合物的后修饰酶中相邻Met 255和Trp 107侧链的交联而被替换。
A transient tyrosyl-like radical with a narrow doublet X-band EPR signal is present during catalase turnover byMycobacterium tuberculosiscatalase-peroxidase (KatG). Labeling of KatG with β-methylene-deuterated tyrosine causes a collapse of the doublet to a singlet, while for 3,5-ring-deuterated tyrosine-labeled enzyme, no changes occur in the EPR signal. Except for the replacement Tyr229Phe, all other single-tyrosine mutants of KatG exhibit the same narrow doublet EPR signal and catalase activity similar to that of the wild-type enzyme. These findings confirm that this catalytically competent radical is associated with Tyr229, whose 3′ and 5′ protons are replaced as a result of cross-links with neighboring Met255 and Trp107 side chains in the post-translationally modified enzyme containing a distal-side Met255-Tyr229-Trp107 adduct.