Identification and characterization of tyrosyl radical formation in Mycobacterium tuberculosis catalase-peroxidase (KatG)
Identification and characterization of tyrosyl radical formation in Mycobacterium tuberculosis catalase-peroxidase (KatG)
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DOI：
10.1074/jbc.m207916200
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发表时间：
2002-11-08
影响因子：
4.8
通讯作者：
Magliozzo, RS
中科院分区：
文献类型：
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作者：
Chouchane, S;Girotto, S;Magliozzo, RS
采用快速冷冻淬灭电子顺磁共振（RFQ-EPR）技术研究了结核分枝杆菌过氧化氢酶-过氧化物酶（KatG）的催化功能及其在抗结核抗生素异烟肼活化中的作用。使用x带EPR在77 K下跟踪KatG与过氧乙酸的反应随时间的变化。在混合后的6.4 ms内以及在经过数百毫秒的时间点处出现双峰EPR信号。此后，单线态信号发展，并最终在1秒后占主导地位，与自由基的总产率类似于0.5自旋/血红素。模拟的光谱提供的EPR参数与酪氨酰自由基。L-3，3-[H-2（2）]酪氨酸标记的KatG的超精细分裂和/或谱线宽度的变化，而L-2，4，5，6，7-[H-2（5）] Dahan标记的KatG的超精细分裂和/或谱线宽度的变化证实了这一分配。自由基形成的初始速率不变，使用3倍或10倍过量的过氧乙酸，与涉及中间体的速率决定步骤一致。虽然化合物I可能是KatG中酪氨酰基自由基的前体，但在自由基形成期间既没有观察到其EPR信号也没有观察到其还原成化合物II。酪氨酰自由基的双重信号被迅速淬灭，通过添加异烟肼和苯甲酰肼，但不是通过异丙肼，它结合不良KatG。
The catalytic function of Mycobacterium tuberculosis catalase-peroxidase (KatG) and its role in activation of the anti-tuberculosis antibiotic isoniazid were investigated using rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) experiments. The reaction of KatG with peroxyacetic acid was followed as a function of time using x-band EPR at 77 K. A doublet EPR signal appears within 6.4 ms after mixing and at time points through hundreds of milliseconds. Thereafter, a singlet signal develops and finally predominates after 1 s, with a total yield of radical similar to0.5 spin/heme. Simulation of the spectra provided EPR parameters consistent with those for tyrosyl radicals. Changes in the hyperfine splitting and/or line width in spectra for L-3,3-[H-2(2)]tyrosine-labeled, but not L-2,4,5,6,7-[H-2(5)]tryptophan-labeled KatG confirmed this assignment. The initial rate of radical formation was unchanged using a 3-fold or 10-fold excess of peroxyacetic acid, consistent with a rate-determining step involving an intermediate. Although Compound I is likely to be the precursor of tyrosyl radical in KatG, neither its EPR signal nor its reduction to Compound II during formation of the radical(s) could be observed. The tyrosyl radical doublet signal was rapidly quenched by addition of isoniazid and benzoic hydrazide, but not by iproniazid, which binds poorly to KatG.