Frech C, Schmid F X
Biochemisches Laboratorium, Universität Bayreuth, Federal Republic of Germany.
J Biol Chem. 1995 Mar 10;270(10):5367-74. doi: 10.1074/jbc.270.10.5367.
The interrelationship between the acquisition of ordered structure, prolyl isomerization, and the formation of the disulfide bonds in assisted protein folding was investigated by using a variant of ribonuclease T1 (C2S/C10N-RNase T1) with a single disulfide bond and two cis-prolyl bonds as a model protein. The thiol-disulfide oxidoreductase DsbA served as the oxidant for forming the disulfide bond and prolyl isomerase A as the catalyst of prolyl isomerization. Both enzymes are from the periplasm of Escherichia coli. Reduced C2S/C10N-RNase T1 is unfolded in 0 M NaCl, but native-like folded in > or = 2 M NaCl. Oxidation of 5 microM C2S/C10N-RNase T1 by 8 microM DsbA (at pH 7.0, 25 degrees C) is very rapid with a t1/2 of about 10 s (the second-order rate constant is 7 x 10(3) s-1 M-1), irrespective of whether the reduced molecules are unfolded or folded. When they are folded, the product of oxidation is the native protein. When they are denatured, first the disulfide bond is formed in the unfolded protein chains and then the native structure is acquired. This slow reaction is limited in rate by prolyl isomerization and catalyzed by prolyl isomerase. The efficiency of this catalysis is strongly decreased by the presence of the disulfide bond. Apparently, the rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease T1. Such a degeneracy could generally be an advantage for protein folding both in vitro and in vivo.
以具有单个二硫键和两个顺式脯氨酰键的核糖核酸酶T1变体（C2S/C10N-RNase T1）作为模型蛋白，研究了有序结构的获得、脯氨酰异构化和辅助蛋白折叠中二硫键形成之间的相互关系。硫醇-二硫键氧化还原酶DsbA作为形成二硫键的氧化剂，脯氨酰异构酶A作为脯氨酰异构化的催化剂。这两种酶均来自大肠杆菌周质。还原型C2S/C10N-RNase T1在0 M NaCl中未折叠，但在≥2 M NaCl中呈天然样折叠。8 μM DsbA（在pH 7.0、25℃）对5 μM C2S/C10N-RNase T1的氧化非常迅速，t1/2约为10 s（二级速率常数为7×10³ s⁻¹ M⁻¹），无论还原分子是未折叠还是折叠状态。当它们折叠时，氧化产物是天然蛋白。当它们变性时，首先在未折叠的蛋白链中形成二硫键，然后获得天然结构。这个缓慢的反应受脯氨酰异构化的速率限制，并由脯氨酰异构酶催化。二硫键的存在会大大降低这种催化效率。显然，在核糖核酸酶T1的氧化折叠中，链折叠、脯氨酰异构化和二硫键形成的顺序可能会有所不同。这种简并性通常在体外和体内的蛋白质折叠中都是一个优势。
