色氨酸 2,3-双加氧酶催化三元复合物的动力学和光谱表征。,Biochemistry

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色氨酸 2,3-双加氧酶催化三元复合物的动力学和光谱表征。
Biochemistry ( IF 2.9 ) Pub Date : 2020-07-13 , DOI: 10.1021/acs.biochem.0c00179
Jiafeng Geng ₁ , Andrew C Weitz ₂ , Kednerlin Dornevil _{1,

3} , Michael P Hendrich ₂ , Aimin Liu _{1,

3}

Affiliation

l-色氨酸( l -Trp) 降解犬尿氨酸途径的第一步由血红素依赖性双加氧酶、色氨酸 2,3-双加氧酶 (TDO) 和吲哚胺 2,3-双加氧酶催化。在这项工作中，我们采用停流光吸收光谱研究了铜质金银菌TDO (cmTDO)的 Michaelis 络合物的动力学行为，以提高我们对氧活化和l- Trp 初始氧化的理解。在停流结果的基础上，进行快速冷冻淬火 (RFQ) 实验以通过穆斯堡尔光谱捕获和表征该中间体。通过结合亚氯酸盐歧化酶-亚氯酸盐系统来产生高浓度的溶解 O ₂，我们能够以几乎定量的产率捕获 cmTDO 的 Michaelis 复合物。RFQ-Mössbauer 结果证实了 Michaelis 配合物是 O ₂结合的亚铁物种。他们揭示了 Michaelis 复合物的电子特性与肌红蛋白的 O ₂加合物的电子特性之间的显着相似性。我们还发现，这种反应中间体的衰变是催化反应的限速步骤。用观察到的逆α-副衬底动力学同位素效应ķ _ħ / ķ _d 0.87±0.03时（吲哚d ₅ - ）升-Trp用作底物。这项工作为细菌 TDO 的 Michaelis 复合物的化学特性提供了重要的光谱证据。

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Kinetic and Spectroscopic Characterization of the Catalytic Ternary Complex of Tryptophan 2,3-Dioxygenase.

The first step of the kynurenine pathway for l-tryptophan (l-Trp) degradation is catalyzed by heme-dependent dioxygenases, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase. In this work, we employed stopped-flow optical absorption spectroscopy to study the kinetic behavior of the Michaelis complex of Cupriavidus metallidurans TDO (cmTDO) to improve our understanding of oxygen activation and initial oxidation of l-Trp. On the basis of the stopped-flow results, rapid freeze-quench (RFQ) experiments were performed to capture and characterize this intermediate by Mössbauer spectroscopy. By incorporating the chlorite dismutase–chlorite system to produce high concentrations of solubilized O₂, we were able to capture the Michaelis complex of cmTDO in a nearly quantitative yield. The RFQ–Mössbauer results confirmed the identity of the Michaelis complex as an O₂-bound ferrous species. They revealed remarkable similarities between the electronic properties of the Michaelis complex and those of the O₂ adduct of myoglobin. We also found that the decay of this reactive intermediate is the rate-limiting step of the catalytic reaction. An inverse α-secondary substrate kinetic isotope effect was observed with a k_H/k_D of 0.87 ± 0.03 when (indole-d₅)-l-Trp was employed as the substrate. This work provides an important piece of spectroscopic evidence of the chemical identity of the Michaelis complex of bacterial TDO.

更新日期：2020-08-04

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