McEvoy M, Lantz C, Lunn C A, Pigiet V
J Biol Chem. 1981 Jul 10;256(13):6646-50.
An altered form of thioredoxin, composed of two peptides with molecular weights of 7 and 5 X 10(3) has been isolated from Escherichia coli B after chromatography on Ultrogel AcA54. The position of the clip site, determined by amino acid sequencing to lie between Pro(64) and Gly(65), is consistent with a cleavage within the hinge region connecting the two prominent folding domains of thioredoxin. The NH2-terminal domain containing the active site, and the carboxyl-terminal domain correspond to the 7 and 5 X 10(3) fragments, respectively. The two peptides combine to form a tight complex, T'12. At concentrations below 10(-6) M, the clipped species was not efficiently reduced by thioredoxin reductase, showing only half the activity of native thioredoxin at a protein concentration of 9 X 10(-8) M. The Km of the clipped species for thioredoxin reductase (2.9 X 10(-6) M) was nearly equivalent to that of native thioredoxin (2.2 X 10(-6) M), and the Vmax for both native and clipped thioredoxins were nearly identical. These data are taken to imply a reversible association of the fragments to produce a thioredoxin equivalent to the intact species in its interaction with thioredoxin reductase. Some evidence exists for an unfolding of the 7 X 10(3) fragment upon dissociation of the complex. A functional role for the two-domain structure of thioredoxin is proposed in which the carboxyl domain serves to stabilize the active site within the amino domain and confer the species specificity in the binding of thioredoxin to thioredoxin reductase.
一种由分子量分别为7×10³和5×10³的两种肽组成的硫氧还蛋白变体，在经Ultrogel AcA54柱层析后从大肠杆菌B中分离得到。通过氨基酸测序确定，剪接位点位于Pro(64)和Gly(65)之间，这与硫氧还蛋白两个主要折叠结构域之间的铰链区发生切割一致。含有活性位点的NH₂末端结构域和羧基末端结构域分别对应于7×10³和5×10³片段。这两种肽结合形成紧密复合物T'12。在浓度低于10⁻⁶ M时，剪接后的物种不能被硫氧还蛋白还原酶有效还原，在蛋白质浓度为9×10⁻⁸ M时，其活性仅为天然硫氧还蛋白的一半。剪接后的物种对硫氧还蛋白还原酶的Km（2.9×10⁻⁶ M）几乎等同于天然硫氧还蛋白的Km（2.2×10⁻⁶ M），并且天然和剪接后的硫氧还蛋白的Vmax几乎相同。这些数据意味着片段之间存在可逆结合，从而产生一种在与硫氧还蛋白还原酶相互作用时等同于完整物种的硫氧还蛋白。有一些证据表明，复合物解离后7×10³片段会发生去折叠。有人提出了硫氧还蛋白两结构域结构的功能作用，其中羧基结构域用于稳定氨基结构域内的活性位点，并赋予硫氧还蛋白与硫氧还蛋白还原酶结合时的物种特异性。
