Ruoppolo M, Lundström-Ljung J, Talamo F, Pucci P, Marino G
Dipartimento di Chimica, Università degli Studi di Salerno, Italy.
Biochemistry. 1997 Oct 7;36(40):12259-67. doi: 10.1021/bi970851s.
Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase A (rd-RNase A) and mixed disulfide between glutathione and reduced RNase A derivative (GS-RNase A) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ESMS) time-course analysis and recovery of the native catalytic activity. The ESMS analysis permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding of rd-RNase A and GS-RNase A was also performed in the presence of glutaredoxin (Grx) and/or protein disulfide isomerase (PDI). All the analyses indicate a pathway of sequential reactions in the formation of native RNase A. First, the reduced protein reacts with a single glutathione molecule to form a mixed disulfide which then evolves to an intramolecular S-S bond via thiol-disulfide exchange. Only at this stage, the intermediate containing one intramolecular S-S reacts with a further glutathione molecule, reiterating the process. An analogous mechanism occurs in the refolding of GS-RNase A. The structural analysis of the intermediates formed during the refolding of RNase A showed for the first time that Grx is actually able to catalyze both formation and reduction of mixed disulfides involving glutatione. In both refolding processes, starting from either rd-RNase A or GS-RNase A, Grx displays a significant catalysis at the early stages of the process. Addition of PDI led to a net catalysis of the entire process without appearing to alter the refolding pathway. In the presence of both Grx and PDI, the two enzymes showed a synergistic activity either starting from rd-RNase A, as previously reported [Lundström, J., and Holmgren, A. (1995) J. Biol. Chem. 270, 7822-7828], or starting from GS-RNase A. Present data suggest that the synergistic effect can be explained assuming that Grx actually facilitates PDI action by catalyzing formation or reduction of mixed disulfides. The mixed disulfides are then rapidly converted into intramolecular disulfides in the presence of PDI. These steps are repeated sequentially throughout the whole refolding, resulting in an immediate formation of fully oxidized species even at the very beginning of the reaction. Finally, a Grx mutant, C14S Grx, in which one of the active site cysteine residues (Cys14) had been replaced by serine, had a similar effect on the distribution of folding intermediates, compared to the wild-type protein, thus demonstrating that Grx acts by a monothiol mechanism either in the reduction or in the oxidation step.
以还原变性的核糖核酸酶A（rd-RNase A）以及谷胱甘肽与还原型核糖核酸酶A衍生物之间的混合二硫键（GS-RNase A）为起始原料，对与二硫键氧化和异构化相关的蛋白质折叠进行了研究。通过添加游离谷胱甘肽（GSH + GSSG）引发折叠，并通过电喷雾质谱（ESMS）时间进程分析和天然催化活性的恢复进行监测。ESMS分析允许对重折叠过程中存在的中间体群体进行鉴定和定量。rd-RNase A和GS-RNase A的重折叠也在谷氧还蛋白（Grx）和/或蛋白质二硫键异构酶（PDI）存在的情况下进行。所有分析均表明天然核糖核酸酶A形成过程中存在一系列连续反应的途径。首先，还原型蛋白质与单个谷胱甘肽分子反应形成混合二硫键，然后通过硫醇-二硫键交换演变为分子内S-S键。只有在这个阶段，含有一个分子内S-S的中间体才会与另一个谷胱甘肽分子反应，重复这个过程。GS-RNase A的重折叠也发生类似的机制。核糖核酸酶A重折叠过程中形成的中间体的结构分析首次表明，Grx实际上能够催化涉及谷胱甘肽的混合二硫键的形成和还原。在从rd-RNase A或GS-RNase A开始的两个重折叠过程中，Grx在过程的早期阶段都表现出显著的催化作用。添加PDI导致整个过程的净催化作用，而似乎没有改变重折叠途径。在同时存在Grx和PDI的情况下，这两种酶从rd-RNase A开始（如先前报道[伦德斯特伦，J., 和霍尔姆格伦，A. (1995) J. Biol. Chem. 270, 7822 - 7828]）或从GS-RNase A开始都表现出协同活性。目前的数据表明，假设Grx实际上通过催化混合二硫键的形成或还原促进PDI的作用，那么协同效应可以得到解释。然后在PDI存在的情况下，混合二硫键迅速转化为分子内二硫键。这些步骤在整个重折叠过程中依次重复，即使在反应刚开始时也能立即形成完全氧化的物种。最后，一个Grx突变体C14S Grx，其中一个活性位点半胱氨酸残基（Cys14）被丝氨酸取代，与野生型蛋白相比，对折叠中间体的分布有类似的影响，从而证明Grx在还原或氧化步骤中通过单硫醇机制起作用。
