Computational and mutational analysis of human glutaredoxin (thioltransferase):: Probing the molecular basis of the low pKa of cysteine 22 and its role in catalysis
Computational and mutational analysis of human glutaredoxin (thioltransferase):: Probing the molecular basis of the low pKa of cysteine 22 and its role in catalysis
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DOI：
10.1021/bi0516327
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发表时间：
2006-04-18
期刊：
影响因子：
2.9
通讯作者：
Mieyal, JJ
中科院分区：
文献类型：
--
作者：
Jao, SC;Ospina, SME;Mieyal, JJ
人谷氧还蛋白（GRx），也称为巯基转移酶，是一种12 kDa的巯基-二硫化物氧化还原酶，对还原含谷胱甘肽的混合二硫化物具有高度选择性。活性位点Cys 22残基的表观pK（a）约为3.5。先前我们观察到，谷氧还蛋白的催化增强作用可完全归因于其Cys 22硫醇部分的pK（a）与产物硫醇的pK（a）之间的差异，各自分别在酶促和非酶促反应中充当离去基团[Srinivasan等人（1997），Biochemistry 36，3199-3206]。连续体静电计算表明，低pKa的Cys 22的结果主要是从稳定的硫醇阴离子通过特定的离子配对与带正电的Lys 19残基，虽然氢键与Thr 21的相互作用也出现贡献。考虑Lys 19的变体以进一步评估Lys 19对Cys 22的pKa的预测作用。通过分子建模生成变体K19 Q和K19 L，并计算每个变体的Cys 22的pK（a）值。对于K19 Q，预测的Cys 22 pK（a）为7.3，而对于K19 L，预测值为8.3。突变对加合的谷胱甘肽部分和GRx之间的相互作用能的影响是从GPx和谷胱甘肽的混合二硫键加合物中谷胱甘肽部分和近端蛋白残基之间的货车德瓦尔斯能和静电能粗略估计的，即，GRx-SSG中间体。与野生型酶中间体的值相比，K19突变体的值仅相差少量。总之，计算分析预测突变酶将具有显著降低的催化速率，同时保留野生型酶所显示的谷胱甘肽特异性。因此，我们构建并表征了两种形式的GRx酶的K19 L和K19 Q突变体。每个突变体保留谷胱甘肽特异性的预测和显示减少的活动，但活动的减少的程度没有预测的理论计算。突变酶的相应Cys 22-巯基pK（a）值的变化，如相应酶的碘乙酰胺失活的pH曲线所示，清楚地表明K19-C22离子对不能完全解释Cys 22巯基的低pK（a）。对Cys 22硫醇盐的稳定的另外贡献可能由Thr 21和相邻α-螺旋的N-末端部分正电荷提供。
Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pKa of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pKa of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GPx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.