Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190.
Probing the catalytic potential of the hamster arylamine N-acetyltransferase 2 catalytic triad by site-directed mutagenesis of the proximal conserved residue, Tyr190.
复制标题
通过近端保守残基 Tyr190 的定点诱变探讨仓鼠芳胺 N-乙酰转移酶 2 催化三联体的催化潜力。
DOI：
10.1111/j.1742-4658.2009.07389.x
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Wagner,CarstonR
中科院分区：
文献类型：
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作者：
Zhou,Xin;Zhang,Naixia;Liu,Li;Walters,KylieJ;Hanna,PatrickE;Wagner,CarstonR
芳香胺N-乙酰转移酶（NATs）在芳香胺和肼类药物的解毒和芳香胺致癌物的活化中起重要作用。由于哺乳动物NAT的催化三联体Cys-His-Asp已被证明对维持蛋白质稳定性至关重要，因此无法评估三联体对催化的改变，我们探索了高度保守的近端残基Tyr 190的影响，Tyr 190与三联体残基之一Asp 122形成直接氢键相互作用，以及与活性位点His 107的潜在π-π堆积相互作用。用Phe、Ile或Ala替换仓鼠NAT 2 Tyr 190耐受性良好，并且未导致蛋白质的总体折叠发生显著改变。然而，停流和稳态动力学分析显示，与野生型相比，Tyr 190对于最大化NAT 2的乙酰化速率和对氨基苯甲酸的转乙酰化速率至关重要。Tyr 190还被证明在决定乙酰化过程中活性位点Cys的pKa以及pH与转乙酰化速率曲线方面发挥重要作用。我们假设pH依赖性与活性位点结构的全局变化相关，这通过野生型和Y190 A的[1H，15 N]杂原子单量子相干光谱的叠加来揭示。这些结果表明，NAT 2的催化效率部分取决于Tyr 190介导的多个侧链对活性位点的静电势和局部构象的集体影响的能力。
ArylamineN‐acetyltransferases (NATs) play an important role in both the detoxification of arylamine and hydrazine drugs and the activation of arylamine carcinogens. Because the catalytic triad, Cys‐His‐Asp, of mammalian NATs has been shown to be essential for maintaining protein stability, rendering it impossible to assess alterations of the triad on catalysis, we explored the impact of the highly conserved proximal residue, Tyr190, which forms a direct hydrogen bond interaction with one of the triad residues, Asp122, as well as a potential pi‐pi stacking interaction with the active site His107. The replacement of hamster NAT2 Tyr190 by either Phe, Ile or Ala was well tolerated and did not result in significant alterations in the overall fold of the protein. Nevertheless, stopped‐flow and steady‐state kinetic analysis revealed that Tyr190 was critical for maximizing the acetylation rate of NAT2 and the transacetylation rate ofp‐aminobenzoic acid when compared with the wild‐type. Tyr190 was also shown to play an important role in determining the pKaof the active site Cys during acetylation, as well as the pH versus the rate profile for transacetylation. We hypothesized that the pH dependence was associated with global changes in the active site structure, which was revealed by the superposition of [1H,15N] heteronuclear single quantum coherence spectra for the wild‐type and Y190A. These results suggest that NAT2 catalytic efficiency is partially governed by the ability of Tyr190 to mediate the collective impact of multiple side chains on the electrostatic potential and local conformation of the active site.