The reactive form of a C-S bond-cleaving, CO2-fixing flavoenzyme
The reactive form of a C-S bond-cleaving, CO2-fixing flavoenzyme
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DOI：
10.1074/jbc.ra118.005554
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发表时间：
2019-03-29
影响因子：
4.8
通讯作者：
DuBois, Jennifer L.
中科院分区：
文献类型：
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作者：
Streit, Bennett R.;Mattice, Jenna R.;DuBois, Jennifer L.
NADPH:2-酮丙基辅酶M氧化还原酶/羧化酶（2-KPCC）是一种细菌二硫氧化还原酶（DSOR），在该家族中具有独特的催化二氧化碳固定作用。2-KPCC与其他dsor的不同之处在于，它具有取代保守组氨酸的苯丙氨酸，在典型的dsor中，组氨酸对于稳定活性位点的还原反应形式至关重要。在这里，我们使用定点诱变和停止流动动力学，研究了2-KPCC的反应形式及其与自杀底物和CO2的单次转换反应。2-KPCC的还原半反应在动力学和光谱上与典型的DSOR GSH还原酶相似，其中活性位点组氨酸被丙氨酸取代。然而，2-KPCC的还原反应形式与典型的dsor不同。在没有组氨酸的情况下，黄素和二硫基团不再像典型的dsor那样通过共价或电荷转移相互作用偶联。与硫氧还毒素类似，控制反应性的pK(a)在7.5和8.1之间似乎是由于二硫醇的半胱氨酸之间共享一个质子，这有效地稳定了攻击的半胱氨酸硫化物，并使其能够破坏底物的强C-S键。组氨酸的缺乏保护了2-KPCC的反应中间体免受不必要的质子化；然而，如果没有它作为催化酸碱的输入，发生羧基化的氧化半反应非常缓慢，限制了整体反应速率。我们得出结论，DSOR活性位点的质子严格调控支持C-S键的裂解和对CO2固定的选择性。
NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2. The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pK(a) between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.