Determinants of chymotrypsin C cleavage specificity in the calcium-binding loop of human cationic trypsinogen
Determinants of chymotrypsin C cleavage specificity in the calcium-binding loop of human cationic trypsinogen
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DOI：
10.1111/febs.12018
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发表时间：
2012-12-01
期刊：
影响因子：
5.4
通讯作者：
Sahin-Toth, Miklos
中科院分区：
文献类型：
--
作者：
Szabo, Andras;Sahin-Toth, Miklos
与 C 类似的胰腺丝氨酸蛋白酶胰凝乳蛋白酶 (CTRC) 可裂解人阳离子胰蛋白酶原钙结合环中的 Leu81Glu82 肽键，从而促进其降解。这是一种针对胰腺中异位胰蛋白酶原激活的保护机制。在本研究中，我们证明 CTRC 对 Leu81Glu82 肽键的裂解具有高度特异性，其他人胰腺糜蛋白酶（CTRB1、CTRB2 和 CTRL1）和弹性蛋白酶（ELA2A、ELA3A 和 ELA3B）不会催化该反应。为了阐明 CTRC 特异性的机制基础，我们调查了这些胰腺蛋白酶对肽底物的初级 (P1) 切割偏好。我们发现 CTRC 在 P1 Leu 后裂解，其催化效率比其他测试的酶至少高十倍。为了评估扩展的亚位点相互作用，我们将 Ala 突变引入人阳离子胰蛋白酶原的 P3、P1'、P3' 和 P4' 氨基酸位置，其中 P1P1' 对应于 Leu81Glu82。有趣的是，CTRC介导的裂解受到突变E82A的三倍刺激，并且不受突变E79A和N84A的影响，但是所有三种突变都损害了特异性并导致ELA2A的裂解增加。突变 E85A 使 CTRC 裂解减少两倍。值得注意的是，其他胰凝乳蛋白酶和弹性蛋白酶即使发生 L81F 或 L81A 突变也不会裂解人阳离子胰蛋白酶原，这为这些酶引入了有利的 P1 残基。我们得出结论，CTRC 对人阳离子胰蛋白酶原中 Leu81Glu82 肽键的特异性裂解主要是由其对亮氨酰肽键的独特高活性决定的，其中 P1' Glu82、P3' Asn84 和 P4' Glu85 残基充当额外的特异性决定因素。
The pancreatic serine protease chymotrypsin similar to C (CTRC) cleaves the Leu81Glu82 peptide bond in the calcium-binding loop of human cationic trypsinogen and thereby promotes its degradation. This serves as a protective mechanism against ectopic trypsinogen activation in the pancreas. In the present study, we demonstrate that cleavage of the Leu81Glu82 peptide bond by CTRC is highly specific, and other human pancreatic chymotrypsins (CTRB1, CTRB2 and CTRL1) and elastases (ELA2A, ELA3A and ELA3B) do not catalyze this reaction. To elucidate the mechanistic basis for CTRC specificity, we surveyed the primary (P1) cleavage preference of these pancreatic proteases on peptide substrates. We found that CTRC cleaved after a P1 Leu with at least tenfold higher catalytic efficiency than other enzymes tested. To assess extended sub-site interactions, we introduced Ala mutations into human cationic trypsinogen at the P3, P1' P3' and P4' amino acid positions, where P1P1' corresponds to Leu81Glu82. Interestingly, CTRC-mediated cleavage was stimulated threefold by mutation E82A and unaffected by mutations E79A and N84A, but all three mutations compromised specificity and resulted in increased cleavage by ELA2A. Mutation E85A decreased CTRC cleavage by twofold. Remarkably, other chymotrypsins and elastases did not cleave human cationic trypsinogen even with the L81F or L81A mutations, which introduced favorable P1 residues for these enzymes. We conclude that specific cleavage of the Leu81Glu82 peptide bond in human cationic trypsinogen by CTRC is primarily determined by its distinctively high activity on leucyl peptide bonds, with the P1' Glu82, P3' Asn84 and P4' Glu85 residues serving as additional specificity determinants.