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首页> 外文期刊>Journal of Molecular Biology >Hidden allostery in human glutathione transferase P1-1 unveiled by unnatural amino acid substitutions and inhibition studies
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Hidden allostery in human glutathione transferase P1-1 unveiled by unnatural amino acid substitutions and inhibition studies

机译：非天然氨基酸取代和抑制研究揭示了人类谷胱甘肽转移酶P1-1的隐藏变构

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摘要

Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis-Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kcat and 300-fold increased KM GSH values, displays an apparent Hill coefficient of 0.82 ± 0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency kcat/KM GSH to near the wild-type value but still yields Hill coefficients of 0.61 ± 0.08 and 0.86 ± 0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl- substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99 ± 0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.
机译：传统的二聚体人类谷胱甘肽转移酶（GST）P1-1的稳态动力学研究没有揭示与Michaelis-Menten行为的明显偏差。相比之下，在亚基界面中对锁和键基序的关键残基Y50进行改造，揭示了酶的变构性质。低活性突变体Y50C的特征在于kcat降低150倍，KM GSH值提高300倍，其表观希尔系数为0.82±0.22。通过非天然的正丁基或正戊基取代对Y50C的巯基进行化学烷基化，可将催化效率kcat / KM GSH提升至接近野生型值，但希尔系数仍分别为0.61±0.08和0.86±0.1。因此，变构动力学行为不依赖于酶的低活性。另一方面，也显示出高催化效率的S-环丁基甲基取代的Y50C的Hill系数为0.99±0.11，表明亚基界面处结构的细微差异会影响复杂的动力学行为。此外，使用乙炔酸对天然GST P1-1的抑制研究表明，与野生型酶非共价结合的配体也可以引发变构动力学行为。因此，我们得出的结论是，GST P1-1结构具有固有的变构，在某些而非全部环境条件下会变得明显。

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