Oxidative Uncoupling in Cysteine Dioxygenase Is Gated by a Proton-Sensitive Intermediate
Oxidative Uncoupling in Cysteine Dioxygenase Is Gated by a Proton-Sensitive Intermediate
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DOI：
10.1021/bi501241d
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发表时间：
2014-12-09
期刊：
影响因子：
2.9
通讯作者：
Pierce, Brad S.
中科院分区：
文献类型：
--
作者：
Crowell, Joshua K.;Li, Wei;Pierce, Brad S.
半胱氨酸双加氧酶（CDO）是一种非血红素单核铁酶，催化L-半胱氨酸（Cys）的O2依赖性氧化产生半胱氨酸亚磺酸（CSA）。这种酶催化Cys catenorase中的第一个承诺步骤;因此，它是哺乳动物硫代谢和氧化还原稳态的核心。具有讽刺意味的是，尽管对CDO的研究持续了近45年，但基本上没有关于其动力学机制的信息。在这项工作中，CDO动力学机制中的化学步骤的时间进行了研究，通过pH/pD依赖的稳态动力学和溶剂同位素效应的k（猫），k（猫）/K-M，（O-2/CSA）耦合。在kcat-pL和k（cat）/KM-pL曲线中分别观察到1.45 +/- 0.05和2.0 +/- 0.1的正常溶剂动力学同位素效应。质子库存实验的PL-独立的区域内（PL 8.5）建议多个溶剂可交换的质子在飞行中的kcat和k（猫）/K-M数据。溶剂粘度的影响也进行了研究，以探测非化学步骤，并验证明显的同位素效应是不是由于增加溶剂粘度的D2 O反应相对于H2O。虽然溶剂粘度确实对kcat和kcat/KM有适度的影响，但该响应不足以解释所观察到的溶剂同位素效应。这表明产物释放对于CDO催化仅是部分限速的。最重要的是，质子库存（O-2/CSA）耦合表明，质子敏感的过渡态直接遵循O-2激活。因此，在Cys氧化之前的瞬时物质的质子化由飞行中的质子门控。这种行为提供了有价值的洞察动力学掩蔽的瞬态催化过程中产生的。
Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine (Cys) to produce cysteine sulfinic acid (CSA). This enzyme catalyzes the first committed step in Cys catabolism; thus, it is central to mammalian sulfur metabolism and redox homeostasis. Ironically, despite nearly 45 years of continued research on CDO, essentially no information has been reported with respect to its kinetic mechanism. In this work, the timing of chemical steps in the CDO kinetic mechanism is investigated by pH/pD-dependent steady-state kinetics and solvent isotope effects on k(cat), k(cat)/K-M, and (O-2/CSA) coupling. Normal solvent kinetic isotope effects of 1.45 +/- 0.05 and 2.0 +/- 0.1 are observed in kcat-pL and k(cat)/KM-pL profiles, respectively. Proton inventory experiments within the pL-independent region (pL 8.5) suggest multiple solvent-exchangeable protons in flight for both kcat and k(cat)/K-M data. The influence of solvent viscosity was also investigated to probe non-chemical steps and to verify that the apparent isotope effects were not attributable to increased solvent viscosity of D2O reactions relative to H2O. Although solvent viscosity did have a modest influence on kcat and kcat/KM, the response is not sufficient to account for the observed solvent isotope effects. This suggests that product release is only partially rate-limiting for CDO catalysis. Most crucially, proton inventory of (O-2/CSA) coupling indicates that a proton-sensitive transition state directly follows O-2 activation. Thus, protonation of a transient species preceding Cys oxidation is gated by protons in flight. This behavior provides valuable insight into the kinetically masked transients generated during catalysis.