Bulaj G, Kortemme T, Goldenberg D P
Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.
Biochemistry. 1998 Jun 23;37(25):8965-72. doi: 10.1021/bi973101r.
Thiol-disulfide exchange reactions are required for many aspects of cellular metabolism including the folding of disulfide-bonded proteins, electron transfer, and numerous regulatory mechanisms. To identify factors influencing the rates of these reactions in polypeptides, the reactivities of Cys thiols in 16 model peptides were measured. For each of the peptides, which contained single Cys residues with thiol pKas ranging from 7.4 to 9.1, the rates of exchange with four disulfide-bonded compounds were measured. In reactions with two of the disulfide reagents, cystine and 2-hydroxyethyl disulfide, the peptide thiols displayed Bronsted correlations between reaction rate and pKa similar to those observed previously with model compounds (betanuc = 0.5 and 0.3, respectively). For two reagents with net charges, oxidized glutathione and cystamine, however, the apparent Bronsted coefficients were 0 and 0.8, respectively. These observations are in striking contrast with those obtained with model compounds, for which the Bronsted coefficients for the nucleophilic thiolates are largely independent of the disulfide-containing compound. The differences in the apparent Bronsted coefficients can be largely accounted for by electrostatic interactions between charged groups on the peptides and disulfide reagents and demonstrate that such interactions can play a dominant role in determining the rates of thiol-disulfide exchange in biological molecules. The results presented here provide an improved basis for predicting the rates of these reactions and suggest ways in which differences in the rates of competing reactions can be either minimized, to simplify the analysis of disulfide-coupled folding reactions, or enhanced, to favor formation of particular disulfides.
硫醇 - 二硫键交换反应在细胞代谢的许多方面都是必需的，包括二硫键结合蛋白的折叠、电子转移以及众多调节机制。为了确定影响多肽中这些反应速率的因素，我们测量了16种模型肽中半胱氨酸硫醇的反应活性。对于每种含有单个半胱氨酸残基且硫醇pKa值在7.4至9.1之间的肽，我们测量了其与四种二硫键结合化合物的交换速率。在与两种二硫试剂胱氨酸和2 - 羟乙基二硫的反应中，肽硫醇的反应速率与pKa之间呈现出布朗斯特相关性，类似于先前在模型化合物中观察到的情况（β值分别为0.5和0.3）。然而，对于另外两种带净电荷的试剂氧化型谷胱甘肽和胱胺，表观布朗斯特系数分别为0和0.8。这些观察结果与在模型化合物中获得的结果形成了鲜明对比，对于模型化合物，亲核硫醇盐的布朗斯特系数在很大程度上与含二硫化合物无关。表观布朗斯特系数的差异在很大程度上可以由肽和二硫试剂上带电基团之间的静电相互作用来解释，这表明这种相互作用在决定生物分子中硫醇 - 二硫键交换速率方面可以发挥主导作用。本文给出的结果为预测这些反应的速率提供了更好的基础，并提出了一些方法，通过这些方法可以将竞争反应速率的差异最小化，以简化二硫键偶联折叠反应的分析，或者增强这种差异，以促进特定二硫键的形成。
