Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase-peroxidase (KatG).
Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase-peroxidase (KatG).
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远端水和残基 315 对配体与结核分枝杆菌过氧化氢酶 (KatG) 结合的影响。
DOI：
10.1021/bi801511u
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发表时间：
2008
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：
Ranguelova,Kalina;Suarez,Javier;Metlitsky,Leonid;Yu,Shengwei;Brejt,ShellyZev;Brejt,SidneyZelig;Zhao,Lin;Schelvis,JohannesPM;Magliozzo,RichardS
结核分枝杆菌（Mtb）的过氧化氢酶-过氧化物酶（KatG）对这种病原体的毒力很重要，也负责异烟肼（INH）的激活，异烟肼是一种用于治疗结核病感染的第一线治疗已有50多年历史的抗生素。过表达的mtkatg含有异质的血红素种群，它们具有不同的光谱特性和功能特性。纯化后在静息酶中积累的六坐标（6-c）血红素被定义为在血红素远端含有弱相关水的独特结构。我们提出了意想不到的发现，这种形式的酶，通常作为少数物种与五坐标（5-c）酶一起存在，是配体结合的有利反应物。使用5-c和6-c形式的不同比例组成的静息酶样品，以及使用在残基315处具有不同倾向稳定6-c形式的替换的KatG突变体，可以证明含有6-c血红素的酶具有更快速的氰化物结合和首选的过氧化物结合。光停止流动和平衡滴定的铁KatG与氰化钾揭示了复杂的行为，部分取决于6-c血红素在静息酶的量。共振拉曼光谱和低温EPR光谱清楚地表明有利于配体（氰化物或过氧化物）与6-c血红素结合。在实验的时间尺度上，5-c和6-c酶形态不平衡。这些结果为特定的水分子可能参与过氧化氢酶-过氧化物酶反应机制的第一阶段提供了证据。
The catalase−peroxidase (KatG) ofMycobacterium tuberculosis(Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. OverexpressedMtbKatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase−peroxidase enzymes.
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影响因子：
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影响因子：
2.9
作者：
Zhao, XB;Yu, H;Magliozzo, RS
通讯作者：
Magliozzo, RS