Cysteine pKa Values for the Bacterial Peroxiredoxin AhpC
Cysteine pKa Values for the Bacterial Peroxiredoxin AhpC
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DOI：
10.1021/bi801718d
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发表时间：
2008-12-02
期刊：
影响因子：
2.9
通讯作者：
Poole, Leslie B.
中科院分区：
文献类型：
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作者：
Nelson, Kimberly J.;Parsonage, Derek;Poole, Leslie B.
鼠伤寒沙门氏菌AhpC是过氧化物氧还蛋白家族的创始成员，过氧化物氧还蛋白家族是一组普遍存在的基于半胱氨酸的过氧化物酶，对过氧化氢、有机氢过氧化物和过氧亚硝酸盐具有高反应性。对于所有的过氧化物氧化还原酶，催化半胱氨酸，称为过氧化物半胱氨酸（C-p），作为亲核试剂攻击过氧化物底物，在活性位点形成半胱氨酸次磺酸。因为硫醇盐是比硫醇基团强得多的亲核试剂，所以通常认为基于半胱氨酸的过氧化物酶应表现出低于8.3-8.5的未扰动值的pK（a）值。在本研究中，使用几种独立的方法来评估AhpC的两个半胱氨酰残基的pK（a）。使用两种不同的碘乙酰胺衍生物的方法对两种半胱氨酸产生了未受干扰的pK（a）值（7.9-8.7），显然是由于与C-p的错误构象的反应性（即，局部展开并从活性位点翻转出来），如X射线晶体学分析所支持的。通过测量AhpC与辣根过氧化物酶对过氧化氢的竞争，获得了5.94 +/- 0.10的功能pK（a），推测反映了完全折叠的活性位点内C-p的滴定;该值与通过分析野生型AhpC的pH依赖性（240）获得的值非常相似（5.84 +/-0.02），并且与从酿酒酵母获得的两种典型的2-半胱氨酸过氧化物氧还蛋白（5.4和6.0）相似。因此，AhpC的pK（a）值平衡了对去质子化硫醇的需要（在pH 7下，类似于90%的C-p将被去质子化）与具有较高pK（a）值的硫醇盐是较强亲核试剂的事实。
Salmonella typhimurium AhpC is a founding member of the peroxiredoxin family, a ubiquitous group of cysteine-based peroxidases with high reactivity toward hydrogen peroxide, organic hydroperoxides, and peroxynitrite. For all of the peroxiredoxins, the catalytic cysteine, referred to as the peroxidatic cysteine (C-p), acts as a nucleophile in attacking the peroxide substrate, forming a cysteine sulfenic acid at the active site. Because thiolates are far stronger nucleophiles than thiol groups, it is generally accepted that cysteine-based peroxidases should exhibit pK(a) values lower than an unperturbed value of 8.3-8.5. In this investigation, several independent approaches were used to assess the pK(a) of the two cysteinyl residues of AhpC. Methods using two different iodoacetamide derivatives yielded unperturbed pK(a) values (7.9-8.7) for both cysteines, apparently due to reactivity with the wrong conformation of C-p (i.e., locally unfolded and flipped out of the active site), as supported by X-ray crystallographic analyses. A functional pK(a) of 5.94 +/- 0.10 presumably reflecting the titration of C-p within the fully folded active site was obtained by measuring AhpC competition with horseradish peroxidase for hydrogen peroxide; this value is quite similar to that obtained by analyzing the pH dependence of the epsilon(240) of wild-type AhpC (5.84 +/- 0.02) and similar to those obtained for two typical 2-cysteine peroxiredoxins from Saccharomyces cerevisiae (5.4 and 6.0). Thus, the pK(a) value of AhpC balances the need for a deprotonated thiol (at pH 7, similar to 90% of the C-p would be deprotonated) with the fact that thiolates with higher pK(a) values are stronger nucleophiles.