SUBSTRATE-SPECIFICITY OF TRYPSIN INVESTIGATED BY USING A GENETIC SELECTION
SUBSTRATE-SPECIFICITY OF TRYPSIN INVESTIGATED BY USING A GENETIC SELECTION
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DOI：
10.1073/pnas.87.17.6659
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发表时间：
1990-09-01
影响因子：
11.1
通讯作者：
CRAIK, CS
中科院分区：
文献类型：
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作者：
EVNIN, LB;VASQUEZ, JR;CRAIK, CS
利用寡核苷酸定向突变和遗传选择相结合的方法，研究了大鼠阴离子胰蛋白酶初级底物专一性的结构决定因素。建立了编码胰酶的文库，该文库取代了底物结合口袋底部的第189和190位氨基酸。对文库中的90,000个转化子进行了具有5个数量级蛋白分解活性的动态范围的遗传选择。精氨酸氨解和酯解的快速筛选证实了纯化菌株的活性。以精氨酸肽和赖氨酸肽为底物，对胰酶和15种部分保留功能的突变胰酶进行了鉴定和动力学研究。底物结合口袋中氨基酸的交替排列维持了有效的催化。第189或190位氨基酸的负电荷被证明是高水平催化所必需的。当天冬氨酸残基位于189位时，几种氨基酸可以取代190位的丝氨酸。精氨酸和赖氨酸底物特异性的调节依赖于190位的氨基酸。190位氨基酸侧链对底物特异性的调节作用也反映在自然产生的胰酶同系物的底物结合口袋中。
The structural determinants of the primary substrate specificity of rat anionic trypsin were examined by using oligonucleotide-directed mutagenesis coupled to a genetic selection. A library was created that encoded trypsins substituted at amino acid positions 189 and 190 at the base of the substrate binding pocket. A genetic selection, with a dynamic range of 5 orders of proteolytic activity, was used to search 90,000 transformants of the library. Rapid screening for arginyl amidolysis and esterolysis confirmed the activity of the purified isolates. Trypsin and 15 mutant trypsins with partially preserved function were identified and characterized kinetically on arginyl and lysyl peptide substrates. Alternative arrangements of amino acids in the substrate binding pocket sustained efficient catalysis. A negative charge at amino acid position 189 or 190 was shown to be essential for high-level catalysis. With the favored aspartic acid residue at position 189, several amino acids could replace serine at position 190. Modulation of the specificity for arginine and lysine substrates was shown to depend on the amino acid at position 190. The regulatory effect of the amino acid side chain at position 190 on the substrate specificity is also reflected in substrate binding pockets of naturally occurring trypsin homologs.