Peroxiredoxin亚磺酰化机理中关键构象转变的动力学。,ACS Catalysis

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Peroxiredoxin亚磺酰化机理中关键构象转变的动力学。
ACS Catalysis ( IF 11.3 ) Pub Date : 2020-01-31 , DOI: 10.1021/acscatal.9b04471
Alexandre Kriznik _{1,

2} , Marouane Libiad ₃ , Hélène Le Cordier ₁ , Samia Boukhenouna ₁ , Michel B Toledano ₃ , Sophie Rahuel-Clermont _{1,

2}

Affiliation

来自Prx1子家族（Prx）的过氧化物酶是月光下的过氧化物酶，在过氧化物信号中起作用，并受亚磺酰化作用调节。Prxs提供了蛋白质-硫醇氧化修饰的主要模型。它们与H2O2反应形成亚磺酸中间体，该亚磺酸中间体参与二硫键，使酶进入其过氧化物酶循环，或再次与过氧化物反应生成亚磺酸，从而使酶失活。亚磺酰化的敏感性取决于这两个竞争反应的动力学，并受到从完全折叠（FF）到局部展开（LU）构象的结构转变的严重影响。通过基于Trp荧光的快速动力学对Tsa1啤酒酵母Prx与H2O2的反应进行分析，发现与FF / LU过渡相关的过程在动力学上不同于二硫化物的形成，并表明亚硫酸盐的形成有助于局部展开。使用具有不同敏感性和不同过氧化物底物的突变体表明，亚磺酰化敏感性与拆分步骤动力学无关，而仅取决于亚磺酸氧化和FF-LU跃迁速率常数。此外，活性位点FF构象的稳定（亚磺酰化动力学的决定因素）仅受确定FF→LU动力学的Prx C末端尾部动力学的影响。根据这两个参数，

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Dynamics of a Key Conformational Transition in the Mechanism of Peroxiredoxin Sulfinylation.

Peroxiredoxins from the Prx1 subfamily (Prx) are moonlighting peroxidases that operate in peroxide signaling and are regulated by sulfinylation. Prxs offer a major model of protein-thiol oxidative modification. They react with H2O2 to form a sulfenic acid intermediate that either engages into a disulfide bond, committing the enzyme into its peroxidase cycle, or again reacts with peroxide to produce a sulfinic acid that inactivates the enzyme. Sensitivity to sulfinylation depends on the kinetics of these two competing reactions and is critically influenced by a structural transition from a fully folded (FF) to locally unfolded (LU) conformation. Analysis of the reaction of the Tsa1 Saccharomyces cerevisiae Prx with H2O2 by Trp fluorescence-based rapid kinetics revealed a process linked to the FF/LU transition that is kinetically distinct from disulfide formation and suggested that sulfenate formation facilitates local unfolding. Use of mutants of distinctive sensitivities and of different peroxide substrates showed that sulfinylation sensitivity is not coupled to the resolving step kinetics but depends only on the sulfenic acid oxidation and FF-to-LU transition rate constants. In addition, stabilization of the active site FF conformation, the determinant of sulfinylation kinetics, is only moderately influenced by the Prx C-terminal tail dynamics that determine the FF → LU kinetics. From these two parameters, the relative sensitivities of Prxs toward hyperoxidation with different substrates can be predicted, as confirmed by in vitro and in vivo patterns of sulfinylation.

更新日期：2020-02-24

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