Qin J, Clore G M, Gronenborn A M
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Biochemistry. 1996 Jan 9;35(1):7-13. doi: 10.1021/bi952299h.
The pH dependence of the 13C chemical shifts of the side-chain carboxyl carbons of all Asp and Glu residues in the reduced and oxidized states of human thioredoxin and in a mixed disulfide complex of human thioredoxin with a target peptide from the transcription factor NF kappa B has been investigated by multidimensional triple-resonance NMR spectroscopy. While the titration curves for most of the side-chain carboxyl resonances exhibit simple Henderson-Hasselbalch behavior with pKa values not far from those found for model compounds, several side chains give rise to two- or three-step titration curves, indicative of the influence of multiple ionizations. In particular, the triad formed by Asp58, Asp60, and Asp61 forms such a complex network of titrating groups. The ionization behavior of Asp26 shows an abnormally high pKa value for an aspartate residue in all states of human thioredoxin, with pKa values of 9.9 in the reduced state, 8.1 in the oxidized state, 8.9 in the mixed disulfide complex, and 8.6 in an active site mutant in which Cys35 was replaced by Ala. The unambiguous determination of the pKa values of Asp26 for a variety of states of human thioredoxin presented in this paper is highly significant in view of two recent reports on Escherichia coli thioredoxin which presented contradicting pKa values for Asp26 and Cys35 [Wilson et al. (1995) Biochemistry 34, 8931-8939; Jeng et al. (1995) Biochemistry 34, 10101-10105]. The stabilization of the protonated side chain of Asp26 in human thioredoxin is achieved via a hydrogen-bonding network involving the hydroxyl group of the neighboring Ser28 which is then connected to the active site region (comprising Cys32 and Cys35) via bound water molecules. The coupling of the buried Asp26 to the active site is responsible for the influence of the Asp26 ionization behavior on the titration shifts of active site residues.
通过多维三重共振核磁共振光谱法，研究了人硫氧还蛋白还原态和氧化态以及人硫氧还蛋白与转录因子NF-κB的靶肽形成的混合二硫键复合物中，所有天冬氨酸（Asp）和谷氨酸（Glu）残基侧链羧基碳的¹³C化学位移对pH的依赖性。虽然大多数侧链羧基共振的滴定曲线呈现简单的亨德森-哈塞尔巴尔赫行为，其pKa值与模型化合物的pKa值相差不远，但有几个侧链产生了两步或三步滴定曲线，这表明存在多个电离的影响。特别是，由Asp58、Asp60和Asp61形成的三联体构成了这样一个复杂的滴定基团网络。在人硫氧还蛋白的所有状态下，Asp26的电离行为显示出天冬氨酸残基异常高的pKa值，还原态下为9.9，氧化态下为8.1，混合二硫键复合物中为8.9，在Cys35被丙氨酸取代的活性位点突变体中为8.6。鉴于最近两篇关于大肠杆菌硫氧还蛋白的报道给出了Asp26和Cys35相互矛盾的pKa值[Wilson等人（1995年），《生物化学》34卷，8931 - 8939页；Jeng等人（1995年），《生物化学》34卷，10101 - 10105页]，本文中明确测定人硫氧还蛋白多种状态下Asp26的pKa值具有重要意义。人硫氧还蛋白中Asp26质子化侧链的稳定是通过一个氢键网络实现的，该网络涉及相邻Ser28的羟基，然后通过结合水分子与活性位点区域（包括Cys32和Cys35）相连。埋藏的Asp26与活性位点的耦合导致了Asp26电离行为对活性位点残基滴定位移的影响。
