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首页> 外文期刊>Biochemistry >Analysis of the link between enzymatic activity and oligomeric state in AhpC, a bacterial peroxiredoxin
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Analysis of the link between enzymatic activity and oligomeric state in AhpC, a bacterial peroxiredoxin

机译：细菌过氧化物酶AhpC中酶活性与寡聚状态之间的联系分析

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Peroxiredoxins (Prxs) make up a ubiquitous class (proposed EC 1.11.1.15) of cysteine-dependent peroxidases with roles in oxidant protection and signal transduction. An intriguing biophysical property of typical 2-Cys Prxs is the redox-dependent modulation of their oligomeric state between decamers and dimers at physiological concentrations. The functional consequences of this linkage are unknown, but on the basis of structural considerations, we hypothesized that decamer-building (dieter-dieter) interactions serve to stabilize a loop that forms the peroxidatic active site. Here, we address this important issue by studying mutations of Thr77 at the decamer-building interface of AhpC from Salmonella typhimurium. Ultracentrifugation studies revealed that two of the substitutions (T771 and T77D) successfully disrupted the decamer, while the third (T77V) actually enhanced decamer stability. Crystal structures of the decameric forms of all three mutant proteins provide a rationale for their properties. A new assay allowed the first ever measurement of the true k(cat) and K-m values of wild-type AhpC with H2O2, placing the catalytic efficiency at 4 x 10(7) M-1 s(-1). T77V had slightly higher activity than wild-type enzyme, and both T77I and T77D exhibited ca. 100-fold lower catalytic efficiency, indicating that the decameric structure is quite important for, but not essential to, activity. The interplay between decamer formation and active site loop dynamics is emphasized by a decreased susceptibility of T77I and T77D to peroxide-mediated inactivation, and by an increase in the crystallographic beta-factors in the active site loop, rather than at the site of the mutation, in the T77D variant.
机译：过氧化物酶（Prxs）构成了普遍存在的半胱氨酸依赖性过氧化物酶类（提议的EC 1.11.1.15），在氧化保护和信号转导中起作用。典型的2-Cys Prxs的一个有趣的生物物理特性是在生理浓度下，十聚体和二聚体之间低聚状态的氧化还原依赖性调节。这种联系的功能后果是未知的，但基于结构上的考虑，我们假设decamer-build（dieter-dieter）相互作用可稳定形成过氧化物活性位点的环。在这里，我们通过研究鼠伤寒沙门氏菌AhpC的十聚体构建界面处的Thr77突变来解决这个重要问题。超速离心研究表明，其中两个取代（T771和T77D）成功破坏了十聚体，而第三个（T77V）实际上增强了十聚体的稳定性。所有三种突变蛋白的十聚体形式的晶体结构为其性质提供了理论依据。一种新的测定方法允许首次用H2O2测量野生型AhpC的真实k（cat）和K-m值，将催化效率设为4 x 10（7）M-1 s（-1）。 T77V具有比野生型酶稍高的活性，并且T77I和T77D都显示约ca。催化效率降低100倍，表明十聚体结构对于活性而言非常重要，但并非必需。 T77I和T77D对过氧化物介导的失活的敏感性降低，以及活性位点环中而不是突变位点处的结晶β因子增加，从而强调了decamer形成与活性位点环动力学之间的相互作用。 ，在T77D变体中。

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