REDESIGNING TRYPSIN - ALTERATION OF SUBSTRATE-SPECIFICITY
REDESIGNING TRYPSIN - ALTERATION OF SUBSTRATE-SPECIFICITY
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DOI：
10.1126/science.3838593
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发表时间：
1985-01-01
期刊：
影响因子：
56.9
通讯作者：
RUTTER, WJ
中科院分区：
文献类型：
--
作者：
CRAIK, CS;LARGMAN, C;RUTTER, WJ
为了研究蛋白水解酶胰蛋白酶的结构与功能之间的关系，发展了一种通过定点突变修饰真核基因并随后在哺乳动物细胞中表达的通用方法。胰蛋白酶结合腔中216和226位的甘氨酸残基被丙氨酸残基取代，产生三个胰蛋白酶突变体。计算机图形分析表明，这些取代将差异影响精氨酸和赖氨酸底物结合的酶。虽然突变酶的催化速率降低，但它们显示出相对于天然酶增强的底物特异性。这种增加的特异性是通过对精氨酸和赖氨酸底物的催化活性的意外差异效应实现的。在位置226处含有丙氨酸的突变体表现出改变的构象，其可以在与底物类似物结合时转化为胰蛋白酶样结构。
A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.