Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
Chem Res Toxicol. 2011 Apr 18;24(4):434-50. doi: 10.1021/tx100413v. Epub 2011 Mar 10.
Protein thiol reactivity generally involves the nucleophilic attack of the thiolate on an electrophile. A low pK(a) means higher availability of the thiolate at neutral pH but often a lower nucleophilicity. Protein structural factors contribute to increasing the reactivity of the thiol in very specific reactions, but these factors do not provide an indiscriminate augmentation in general reactivity. Notably, reduction of hydroperoxides by the catalytic cysteine of peroxiredoxins can achieve extraordinary reaction rates relative to free cysteine. The discussion of this catalytic efficiency has centered in the stabilization of the thiolate as a way to increase nucleophilicity. Such stabilization originates from electrostatic and polar interactions of the catalytic cysteine with the protein environment. We propose that the set of interactions is better described as a means of stabilizing the anionic transition state of the reaction. The enhanced acidity of the critical cysteine is concurrent but not the cause of catalytic efficiency. Protein stabilization of the transition state is achieved by (a) a relatively static charge distribution around the cysteine that includes a conserved arginine and the N-terminus of an α-helix providing a cationic environment that stabilizes the reacting thiolate, the transition state, and also the anionic leaving group; (b) a dynamic set of polar interactions that stabilize the thiolate in the resting enzyme and contribute to restraining its reactivity in the absence of substrate; but upon peroxide binding these active/binding site groups switch interactions from thiolate to peroxide oxygens, simultaneously increasing the nucleophilicity of the attacking sulfur and facilitating the correct positioning of the substrate. The switching of polar interaction provides further acceleration and, importantly, confers specificity to the thiol reactivity. The extraordinary thiol reactivity and specificity toward H(2)O(2) combined with their ubiquity and abundance place peroxiredoxins, along with glutathione peroxidases, as obligate hydroperoxide cellular sensors.
蛋白质巯基反应性通常涉及硫醇盐对亲电试剂的亲核攻击。低 pK(a) 意味着在中性 pH 下硫醇盐的可用性更高，但通常亲核性较低。蛋白质结构因素有助于在非常特定的反应中增加巯基的反应性，但这些因素并不能提供一般性反应性的无差别增强。值得注意的是，过氧化物酶中的催化半胱氨酸可以将过氧化物还原为氢过氧化物，相对于游离半胱氨酸可以实现非常高的反应速率。这种催化效率的讨论集中在稳定硫醇盐以增加亲核性上。这种稳定源于催化半胱氨酸与蛋白质环境的静电和极性相互作用。我们提出，这组相互作用可以更好地描述为稳定反应的阴离子过渡态的一种方式。关键半胱氨酸的增强酸度是协同的，但不是催化效率的原因。蛋白质通过以下方式稳定过渡态：(a) 围绕半胱氨酸的相对静态电荷分布，包括保守的精氨酸和α-螺旋的 N 端，提供阳离子环境，稳定反应中的硫醇盐、过渡态，以及阴离子离去基团；(b) 一组动态的极性相互作用，稳定酶中的硫醇盐，并有助于在没有底物的情况下抑制其反应性；但在过氧化物结合后，这些活性/结合位点基团从硫醇盐切换到过氧化物氧，同时增加攻击硫的亲核性，并促进底物的正确定位。极性相互作用的切换提供了进一步的加速，并且重要的是，赋予了巯基反应性特异性。过氧化物酶与谷胱甘肽过氧化物酶一起，由于其广泛存在和丰富性，具有非凡的巯基反应性和对 H(2)O(2)的特异性，成为必需的过氧化氢细胞传感器。
