Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide
Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide
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DOI：
10.1006/jmbi.1999.3089
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发表时间：
1999-09-17
影响因子：
5.6
通讯作者：
Sadler, JE
中科院分区：
文献类型：
--
作者：
Lu, DS;Fütterer, K;Sadler, JE
肠肽酶是一种膜结合丝氨酸蛋白酶，通过切割和激活胰蛋白酶原来启动胰腺水解酶的激活。该酶具有显著的特异性，在类似胰蛋白酶原活化肽（如瓦尔-（Asp）（4）Lys）的肽基底物的赖氨酸残基后裂解。为了表征底物特异性的决定因素，我们解决了牛肠肽酶催化结构域的晶体结构，以2.3埃分辨率与抑制剂瓦尔-（Asp），赖氨酸-氯甲烷复合。在底物位置P1处与赖氨酸的催化机制和接触与其他胰蛋白酶样丝氨酸蛋白酶是保守的。然而，在位置P2-P4的抑制剂的乙酰基残基与酶表面的相互作用，主要是通过与Lys 99的N-ζ原子的盐桥。将Lys 99突变为Ala或用乙酸酐乙酰化，特异性地阻止胰蛋白酶原或Gly-（Asp）（4）-Lys-beta-naphthylamide的裂解，并将瓦尔-（Asp），Lys-chloromethane的抑制率降低22至90倍。对于这些反应，Lys 99计算为过渡态结合自由能的1.8至2.5 kcal mol-1。因此，肠肽酶表面上的一个独特的碱性外位点已经进化，以促进其生理底物胰蛋白酶原的切割。(C)北京：科学出版社.
Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)(4)Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 Angstrom resolution in complex with the inhibitor Val-(Asp),Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the N-zeta; atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)(4)-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp),Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol-l of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen. (C) 1999 Academic Press.