Characterization of the binding of isoniazid and analogues to Mycobacterium tuberculosis catalase-peroxidase.
Characterization of the binding of isoniazid and analogues to Mycobacterium tuberculosis catalase-peroxidase.
复制标题
异烟肼及其类似物与结核分枝杆菌过氧化氢酶-过氧化物酶结合的表征。
DOI：
10.1021/bi062218p
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Magliozzo,RichardS
中科院分区：
文献类型：
--
作者：
Zhao,Xiangbo;Yu,Shengwei;Magliozzo,RichardS
一线抗结核药物异烟肼(INH)是一种前药，其杀菌功能需要结核分枝杆菌唾液酸酶-过氧化物酶(KatG)激活才能产生酰基-NAD加合物。异烟肼的过氧化被认为是药物作用所必需的催化过程。使用光学和量热技术研究了异烟肼和一系列肼类似物与静止的KatG的结合，提供了热力学参数、结合化学计量和动力学常数(开和关速率)。这项工作揭示了这些底物与一小部分六配位的血红素铁的铁酶的高亲和力结合，这一物种最有可能包含一个弱结合的水分子，它在酶的储存过程中积累。酰肼的结合伴随着很大的热损(>；100kcal/−)，离解常数在0.05kJ/μ~(-1)M范围内，光学停流测量表明kon值在0.5kon值~(27×10~3m~(-1)s~(-1))的范围内，而离解速率很小。在5−8的范围内，结合参数不受pH的影响。在两种突变酶中，异烟肼的高亲和力结合被破坏，这两种酶含有关键末端残基的替换，KatG[W107F]和KatG[Y229F]。酰肼还原KatG化合物I的速率与与休眠酶结合的速率平行。在KatG介导的仿生激活实验中，只有异烟肼才能很好地产生被认为是异烟肼作用中的关键分子的酰基-NAD加合物，从而更好地理解异烟肼的作用机制。
The first-line antituberculosis drug isonicotinic hydrazide (INH) is a prodrug whose bactericidal function requires activation byMycobacterium tuberculosiscatalase-peroxidase (KatG) to produce an acyl-NAD adduct. Peroxidation of INH is considered a required catalytic process for drug action. The binding of INH and a series of hydrazide analogues to resting KatG was examined using optical and calorimetric techniques to provide thermodynamic parameters, binding stoichiometries, and kinetic constants (on and off rates). This work revealed high-affinity binding of these substrates to a small fraction of ferric enzyme in a six-coordinate heme iron form, a species most likely containing a weakly bound water molecule, which accumulates during storage of the enzyme. The binding of hydrazides is associated with a large enthalpy loss (>100 kcal/mol); dissociation constants are in the range of 0.05−1.6 μM, and optical stopped-flow measurements demonstratedkonvalues in the range of 0.5−27 × 103M-1s-1with very smallkoffrates. Binding parameters did not depend on pH in the range 5−8. High-affinity binding of INH is disrupted in two mutant enzymes bearing replacements of key distal side residues, KatG[W107F] and KatG[Y229F]. The rates of reduction of KatG Compound I by hydrazides parallel the on rates for association with the resting enzyme. In a KatG-mediated biomimetic activation assay, only isoniazid generated in good yield the acyl-NAD adduct which is considered a key molecule in INH action, providing a better understanding of the action mechanism of INH.