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首页> 外文期刊>The FEBS journal >C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism
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C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism

机译：植物半胱氨酸蛋白酶的C末端延伸通过部分抑制机制调节蛋白水解活性

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摘要

The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases. Kinetic stability analyses (T-50 and t(1/2)) and refolding experiments show similar thermal stability and refolding efficiency for both forms. These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism.
机译：ervatamin-C的氨基酸序列是热带植物的一种热稳定的半胱氨酸蛋白酶，在其C端（CT）处显示了另外的24个氨基酸延伸。据报道，该延伸肽在酶原激活，催化活性，折叠和稳定性中的作用。对于这项研究，我们在大肠杆菌中表达了两种重组形式的蛋白酶，一种保留了CT延伸，另一种被截断了。具有延伸的酶在较高的pH 8.0下显示出自催化酶原活化，而延伸的缺失导致酶的活性更高。没有发现这种CT延伸在自身催化过程中被切割，或者被不同的外部蛋白酶通过有限的蛋白水解而被切割。分子建模和模拟研究表明，CT延伸可阻断一些底物结合性未引发的亚位点，包括酶的特异性确定亚位点（S2），从而部分阻止了底物与活性位点的可及性，这也证实了实验观察。模型结构中的CT延伸显示出与酶催化结构域的紧密堆积，这是由强烈的疏水和H键相互作用所介导的，因此限制了其切割位点对蛋白酶本身或对外部蛋白酶的可及性。动力学稳定性分析（T-50和t（1/2））和复性实验表明两种形式的热稳定性和复性效率相似。这些数据表明，CT延伸在ervatamin-C的蛋白水解活性中具有抑制作用，但在稳定酶或其折叠机制中没有主要作用。

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