Background

M20 aminopeptidases, such as Peptidase T (PepT), are implicated in the hydrolysis of oligopeptides during the terminal stages of protein degradation pathway to maintain turnover. Therefore, specific inhibition of PepT bores well for the development of novel next-generation antileishmanials. This work describes the metal dependence, substrate preferences and inhibition of PepT, and demonstrates in detail the role of its two conserved substrate binding residues.

Methods

PepT was purified and characterized using a scheme of peptide substrates and peptidomimetic inhibitors. Residues T364 and N378 were mutated and characterized with an array of biochemical, biophysical and structural biology methods.

Results

PepT sequence carries conserved motifs typical of M20 peptidases and our work on its biochemistry shows that this cytosolic enzyme carries broad substrate specificity with best cleavage preference for peptides carrying alanine at the P¹ position. Peptidomimetics amastatin and actinonin occupied S¹ pocket by competing with the substrate for binding to active site and inhibited PepT potently, while arphamenine A and bestatin were less effective inhibitors. We further show that the mutation of conserved substrate binding residues (T364 and N378) to alanine affects structure, reduces substrate binding and alters the amidolytic activity of this dimeric enzyme.

Conclusions

PepT preferentially hydrolyzes oligopeptides carrying alanine at P¹ position and is potently inhibited by peptidomimetics. Reduced substrate binding after mutations was a key factor involved in amidolytic digressions.

General significance

This study provides insights for further exploration of the druggability of PepT and highlights prospective applications of this enzyme along with its mutazyme T364A/N378A.

中文翻译：

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利什曼肽酶T的关键底物结合残基的突变改变其功能和结构动力学。

背景

M20氨基肽酶（例如肽酶T（PepT））在蛋白质降解途径的最终阶段涉及寡肽的水解，以维持营业额。因此，对PepT的特异性抑制作用对于新型下一代抗真菌药的开发很有效。这项工作描述了金属依赖性，底物偏好和对PepT的抑制，并详细证明了其两个保守的底物结合残基的作用。

方法

使用肽底物和拟肽抑制剂的方案纯化和鉴定PepT。用一系列生化，生物物理和结构生物学方法对残基T364和N378进行突变和表征。

结果

PepT序列带有M20肽酶典型的保守基序，我们的生物化学工作表明，这种胞质酶具有广泛的底物特异性，对在P ¹位置带有丙氨酸的肽具有最佳的切割偏好。拟肽类阿马他汀和肌动蛋白通过与底物竞争结合到活性位点而占据了S ¹口袋，并有效抑制了PepT，而香菜碱A和Bestatin的抑制作用较差。我们进一步表明，保守的底物结合残基（T364和N378）向丙氨酸的突变影响了结构，降低了底物结合并改变了这种二聚酶的酰胺分解活性。

结论

PepT优先水解在P ¹位置带有丙氨酸的寡肽，并被拟肽类有效抑制。突变后降低的底物结合是酰胺水解离题的关键因素。

一般意义

这项研究为进一步研究PepT的可药用性提供了见识，并着重指出了该酶及其诱变酶T364A / N378A的前瞻性应用。