Aspartic acid 26 in reduced Escherichia coli thioredoxin has a pKa > 9.
Aspartic acid 26 in reduced Escherichia coli thioredoxin has a pKa > 9.
复制标题
还原型大肠杆菌硫氧还蛋白中的天冬氨酸 26 的 pKa > 9。
DOI：
10.1021/bi00028a001
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Woodward,C
中科院分区：
文献类型：
--
作者：
Wilson,NA;Barbar,E;Fuchs,JA;Woodward,C
1995年5月30日收到的修订版Mandarin pt ®摘要：已表征还原硫氧还蛋白中活性位点残基Asp 26、Cys 32和Cys 35的表观pATa值。野生型硫氧还蛋白和突变型D26 A硫氧还蛋白在半胱氨酸/3-碳上选择性富集13 C。在这两种蛋白质中，测量了“HB 1”、“HB 2”和13 CB NMR化学位移随pH的变化。在野生型还原的硫氧还蛋白中，对于两种半胱氨酸，HB 1质子的pH相对于化学位移的图可以拟合为pKd值为7.0-7.1的一次滴定。相比之下，HB 2质子和β-碳给出的pH-化学位移图清楚地反映了不止一次滴定;拟合数据得到HB 2质子的表观pKd值为7.0-7.3和9.5，CB碳的表观pKd值为7.5-7.9和9.2-10.2。在还原的D26 A中，所有三种探针化学位移都具有pH依赖性，其通过pKd为7.4-7.9的一次滴定来拟合。D26 A中不存在PA”a> 9的滴定，加上通过拉曼光谱法测定的半胱氨酸巯基PA”a值为7.1和7.9 [Li等人.（1993）Biochemistry 32，5800-5808]，表明还原的硫氧还蛋白中的pKd> 9是Asp 26的pKd。鉴于先前的观察结果，这是非常重要的，即在氧化的硫氧还蛋白中，Asp 26 pKd为7.5 [Langsetmo et al.（1991）Biochemistry 30，7603-7609]。这些羧基的非常高的pKd值与它们在三维结构中的局部环境一致;氧化的硫氧还蛋白中的Asp 26侧链几乎但不完全被掩埋，并且在还原的硫氧还蛋白中，它可能被甚至更多地掩埋。Asp 26在还原型硫氧还蛋白中的pKa上移解释了还原型硫氧还蛋白的稳定性低于氧化型硫氧还蛋白，并表明这种高度保守的活性位点群的功能是通过热力学联系来调节酶的氧化还原电位以实现整体稳定性。还原型硫氧还蛋白中Asp 26的较高pKd也为噬菌体成熟过程中对还原型硫氧还蛋白的偏好提供了解释。
Revised Manuscript Received May 30, 1995® abstract: Apparent pATa values of active site residuesAsp26, Cys32, and Cys35 in reduced thioredoxin have been characterized. Bothwild-type thioredoxin and mutant D26A thioredoxin were selectively 13C-enriched on cysteine/3-carbons. In both proteins, the variation with pH of'HBl,'HB2, and 13CB NMR chemical shifts has been measured. In wild-type reduced thioredoxin, for both cysteines, the pH versus chemical shift plots of HBl protons can be fit to one titration with pKd values of 7.0—7.1. In contrast, the HB2 protons and/3-carbons give pH—chemical shift plots that clearly reflect more than one titration; fits to the data give apparent pKd values of 7.0—7.3 and 9.5 for HB2 protons and 7.5—7.9 and 9.2—10.2 for CB carbons. In reduced D26A, all three probe chemical shifts have a pH dependence that is fit by one titration with pKd of 7.4—7.9. The absence of a titration with pA" a> 9 in D26A, taken together with cysteine thiol pA" a values of 7.1 and 7.9 determined by Raman spectroscopy [Li et al.(1993) Biochemistry 32, 5800—5808], indicates that the pKd> 9 in reduced thioredoxin is that of Asp26. This is highly significant in view of the previous observation that, in oxidized thioredoxin, Asp26 pKd is 7.5 [Langsetmo et al.(1991) Biochemistry 30, 7603—7609], The very high pKd values of these carboxyls is consistent with their local environment in the three-dimensional structure; the Asp26 side chain in oxidized thioredoxin is almost but not completely buried, and in reduced thioredoxin it may be even more buried. The upward shift in pKa of Asp26 in reduced thioredoxin accounts for the lower stability of reduced compared to oxidized thioredoxin and suggests that the function of this very highly conserved active site group isto regulate the redox potential of the enzyme through thermodynamic linkage to global stability. The higher pKd of Asp26 in reduced thioredoxin also offers an explanation for the preference for reduced thioredoxin in phage maturation processes.