Specificity of trypsin digestion and conformational flexibility at different sites of unfolded lysozyme
Specificity of trypsin digestion and conformational flexibility at different sites of unfolded lysozyme
复制标题
胰蛋白酶消化的特异性和未折叠溶菌酶不同位点的构象灵活性
作者：
Y. Noda;Keiro Fujiwara;Koji Yamamoto;Takuji Fukuno;S. Segawa
14个胰蛋白酶肽和9个中间体被鉴定为还原和S-3-（三甲基氨基）丙基化溶菌酶的胰蛋白酶消化产物。通过监测色谱图上的峰面积观察这些产物出现和消失的动力学。尽管反应途径复杂，但蛋白质和几种中间产物的消化动力学显示出具有单一速率常数的简单衰减曲线。在本文中，胰蛋白酶的敏感性的个别切割位点被定义为在10 nM胰蛋白酶的存在下，敏感的肽键的水解速率常数。根据胰蛋白酶敏感性，未折叠溶菌酶的切割位点分为两组：一组具有高敏感性（10-20 h-1），另一组具有低敏感性（1.0-2.0 h-1）。总之，在溶菌酶的未折叠状态下，从残基15到61的区域对胰蛋白酶消化具有很强的抵抗力;另一方面，多肽链的C末端的一半足够灵活，可以适应胰蛋白酶的活性位点。
Fourteen tryptic peptides and nine intermediates were identified as products of trypsin digestion of reduced and S‐3‐(trimethylated amino) propylated lysozyme. Kinetics of the appearance and disappearance of these products were observed by monitoring the peak areas on the chromatogram. In spite of the complicated reaction pathways, kinetics of the digestion of proteins and several intermediate products show simple decay curves with a single rate constant. In this paper, the trypsin susceptibility of the individual cleavage site is defined as a hydrolytic rate constant of the susceptible peptide bond in the presence of 10 nM trypsin. The cleavage sites of unfolded lysozyme are classified into two groups in terms of the trypsin susceptibility: one has a high susceptibility (10–20 h−1) and the other a low susceptibility (1.0–2.0 h−1). In the unfolded state of lysozyme, in conclusion, the region from residues 15 to 61 has a strong resistance to trypsin digestion; on the other hand, the C‐terminal half of the polypeptide chain is flexible enough to fit into the active site of trypsin.