Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: Structural and functional characterization of mutants of Asp 26 and Lys 57
Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: Structural and functional characterization of mutants of Asp 26 and Lys 57
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DOI：
10.1021/bi961801a
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发表时间：
1997-03-04
期刊：
影响因子：
2.9
通讯作者：
Holmgren, A
中科院分区：
文献类型：
--
作者：
Dyson, HJ;Jeng, MF;Holmgren, A
为了研究Asp 26和Lys 57在大肠杆菌硫氧还蛋白（Trx）氧化还原机制中的作用，制备了Asp 26 → Ala（D26 A）、Lys 57 → Met（K57 M）和双突变体D26 A/K57 M。突变型硫氧还蛋白的氧化（Trx-S-2）和还原[Trx-（SH）（2）]形式均完全折叠，且整体结构与野生型蛋白（wt）相似。活性位点疏水表面的结构未因Asp 26和Lys 57的突变而改变，因为T7基因5蛋白和突变体Trx-（SH）（2）的1：1复合物中的DNA聚合酶活性显示突变体和野生型的K-d值相似（相似于5 nM）。相反，涉及硫氧还蛋白作为二硫化物还原或二硫醇氧化的催化剂的氧化还原反应受到突变的强烈影响。在Trx-S-2与硫氧还蛋白还原酶在pH 8.0下的反应中，D26 A突变体的k（cat）/K-m值从wt的k（cat）/K-m值降低了10倍，而D26 A/K57 M突变体的值降低了40倍。用胰岛素测量Trx-（SH）（2）作为蛋白质二硫键还原酶的活性，使用荧光检测硫氧还蛋白的氧化。在15 ℃和pH 8.0下，D26 A和K57 M突变体的反应速率与野生型相比均显示出5-10倍的降低，并且与wt曲线相比，突变体的pH-速率曲线向更高的pH移动了1（K57 M）和2（D26 A）个单位。三种突变蛋白质的NMR测量表明，蛋白质具有相同的全球折叠的野生型，虽然一些共振的化学位移的变化表明在活性位点区域的局部结构变化。氧化的D26 A和D26 A/K57 M的共振在pH 6.0和10.0之间不依赖于pH，证实了在wt Trx-S-2中pK（a）为7.5的活性位点基团滴定为Asp 26。对于K57 M Trx-S-2，观察到Asp 26的pK（a）发生了显著变化，从野生型的7.5变为突变体的9.4。共振的pH依赖性行为在所有突变体Trx-（SH）（2）蛋白中受到影响。在Cys 32和Cys 35 C β共振上均观察到单个pK（a）偏移至较高值。作为wt Trx-（SH）（2）的pH的函数的紫外吸光度测量（A（240））证明半胱氨酸硫醇滴定具有约7.1和9.9的表观pK（a）s。突变体蛋白各自在A（240）测量中显示出单一转变，其中点在pH 7.8-8.0，与NMR结果一致。在240 nm处的吸光度随pH增加的变化表明，在每个突变体中滴定的硫醇的数量大于1但小于2。很明显，两个巯基pK（a）都因突变而发生显著偏移。Cys 32 pK（a）从wt中的7.1移动到突变体中的7.8-8.0。Cys 35 pK（a）的值与Cys 32的值不可区分，因此，在UV吸光度测量中考虑了多于一个硫醇滴定，否则转移到更高的pH值（>10）其中，在UV和NMR测量中，它的跃迁被酪氨酸残基的电离和蛋白质的解折叠的影响所掩盖。羧基和Lys 57 ε-氨基显著影响活性位点硫醇的pK（a），特别是暴露的低pK（a）硫醇Cys 32的pK（a），从而提高在生理pH下硫醇-二硫化物反应的速率。
To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes. Both the oxidized (Trx-S-2) and reduced [Trx-(SH)(2)] forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt). The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)(2) shows similar K-d values (similar to 5 nM) for both mutants and wt. In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations. In the reaction of Trx-S-2 with thioredoxin reductase at pH 8.0, the k(cat)/K-m value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold. The activity of Trx-(SH)(2) as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin. At 15 degrees C and pH 8.0, both the D26A and K57M mutants showed 5-10-fold decreases in rates of reaction compared to those of the wild type, and the pH-rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve. NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region. The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pK(a) of 7.5 in wt Trx-S-2 as Asp 26. A profound change in the pK(a) of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S-2. The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)(2) proteins. A single pK(a) shifted to higher values is observed on both the Cys 32 and Cys 35 C beta resonances. Ultraviolet absorbance measurements (A(240)) as a function of pH for wt Trx-(SH)(2) demonstrate that the cysteine thiols titrate with apparent pK(a)s of about 7.1 and 9.9. The mutant proteins each show a single transition in the A(240) measurements, with a midpoint at pH 7.8-8.0, consistent with the NMR results. The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two. It is clear that both thiol pK(a)s have been significantly shifted by the mutations. The Cys 32 pK(a) is moved from 7.1 in wt to 7.8-8.0 in the mutants. The value of the Cys 35 pK(a) either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance measurements or else is shifted to much higher pHs (>10) where its transition is masked in both UV and NMR measurements by the effects of ionization of the tyrosine residues and unfolding of the protein.Our results strongly suggest that the buried Asp 26 carboxyl and Lys 57 epsilon-amino groups significantly affect the pK(a)s of the active site thiols, particularly that of the exposed low-pK(a) thiol Cys 32, thereby enhancing the rates of thiol-disulfide reactions at physiological pH.