Stefansson Bjarki, Helgadóttir Linda, Olafsdottir Sigridur, Gudmundsdottir Agústa, Bjarnason Jón B
Department of Biochemistry, Science Institute University of Iceland, Dunhaga 3, 107 Reykjavík, Iceland.
Comp Biochem Physiol B Biochem Mol Biol. 2010 Feb;155(2):186-94. doi: 10.1016/j.cbpb.2009.11.004.
Atlantic cod trypsin I is a highly active cold-adapted protease. This study aimed at further characterization of this enzyme with respect to kinetic parameters, sites of autolysis and stability. For that purpose, trypsin I was purified by anion exchange chromatography. Its purity and identity was verified by SDS-PAGE analysis and mass spectrometry. Concomitantly, another cod trypsin isozyme, trypsin X, previously only described from its cDNA sequence was detected in a separate peak from the ion exchange chromatogram. There was a stepwise increase in the catalytic efficiency (k(cat)/K(m)) of cod trypsin I obtained with substrates containing one to three amino acid residues. As expected, the activity of trypsin I was maintained for longer periods of time at 15 degrees C than at higher temperatures. The residues of the trypsin I molecule most sensitive to autolysis were identified using Edman degradation. Eleven autolytic cleavage sites were detected within the trypsin I molecule. Unfolding experiments demonstrated that autolysis is a contributing factor in the stability of trypsin I. In addition, the data shows that cod trypsin I is less stable towards thermal unfolding than its mesophilic bovine analogue.
大西洋鳕鱼胰蛋白酶I是一种活性很高的冷适应性蛋白酶。本研究旨在进一步表征该酶的动力学参数、自溶位点和稳定性。为此，通过阴离子交换色谱法纯化胰蛋白酶I。通过SDS-PAGE分析和质谱法验证其纯度和同一性。同时，在离子交换色谱图的一个单独峰中检测到另一种鳕鱼胰蛋白酶同工酶，即胰蛋白酶X，此前仅从其cDNA序列中描述过。用含有一至三个氨基酸残基的底物获得的鳕鱼胰蛋白酶I的催化效率（k(cat)/K(m)）呈逐步增加。正如预期的那样，胰蛋白酶I在15℃下的活性比在较高温度下维持的时间更长。使用埃德曼降解法鉴定了胰蛋白酶I分子中对自溶最敏感的残基。在胰蛋白酶I分子内检测到11个自溶切割位点。展开实验表明自溶是胰蛋白酶I稳定性的一个影响因素。此外，数据表明鳕鱼胰蛋白酶I比其嗜温牛胰蛋白酶类似物对热展开更不稳定。
