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首页> 外文期刊>The FEBS journal >Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2' of substrates
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Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2' of substrates

机译：人类肥大细胞糜酶的Arg143和Lys192介导底物P2'位置对酸性氨基酸的偏好

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摘要

Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 -> Gln and Lys192 -> Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.
机译：糜蛋白酶是胰凝乳蛋白酶样丝氨酸蛋白酶，在肥大细胞颗粒中大量发现。到目前为止，已经确定了八个这样的乳糜酶的扩展切割特异性，并且显示了其中四个对P2'位置的酸性氨基酸具有强烈的偏好。这些酶在143和192位分别具有碱性氨基酸（分别为Arg和Lys）。因此，我们假设人糜酶的Arg143和Lys192介导了底物P2'位置对酸性氨基酸的偏好。为了解决这个问题，我们在人糜酶中对这两个位置进行了定点诱变。对两个单一突变体（Arg143-> Gln和Lys192-> Met）和组合的双重突变体的扩展切割特异性的分析显示，其对P2'氨基酸的特异性改变，而所有其他位置基本上不受影响。对于两个单一突变体，观察到对P2'位置的酸性氨基酸的偏爱减弱，而双重突变体缺乏该偏爱。因此，我们得出结论，人糜酶中的位置143和192导致对位置P2'处带负电荷的氨基酸的强烈偏好。这是首次显示在乳糜中，除了位置P1以外，类似的联合效应还影响切割特异性。此外，几种肥大细胞乳糜酶对酸性P2'氨基酸的偏好保留清楚表明，除血管紧张素I以外的其他底物可能是这些酶的主要体内靶标。

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