Craik C S, Largman C, Fletcher T, Roczniak S, Barr P J, Fletterick R, Rutter W J
Science. 1985 Apr 19;228(4697):291-7. doi: 10.1126/science.3838593.
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.
为了研究蛋白水解酶胰蛋白酶的结构与功能之间的关系，开发了一种通过位点特异性诱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。胰蛋白酶结合腔中第216和226位的甘氨酸残基被丙氨酸残基取代，产生了三种胰蛋白酶突变体。计算机图形分析表明，这些取代将对该酶的精氨酸和赖氨酸底物结合产生不同影响。尽管突变酶的催化速率降低，但相对于天然酶，它们表现出增强的底物特异性。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产生意外的差异效应来实现的。在第226位含有丙氨酸的突变体表现出改变的构象，该构象在结合底物类似物后可能转变为胰蛋白酶样结构。
