STRUCTURAL ORIGINS OF SUBSTRATE DISCRIMINATION IN TRYPSIN AND CHYMOTRYPSIN
STRUCTURAL ORIGINS OF SUBSTRATE DISCRIMINATION IN TRYPSIN AND CHYMOTRYPSIN
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DOI：
10.1021/bi00005a004
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发表时间：
1995-02-07
期刊：
影响因子：
2.9
通讯作者：
FLETTERICK, RJ
中科院分区：
文献类型：
--
作者：
PERONA, JJ;HEDSTROM, L;FLETTERICK, RJ
将胰蛋白酶的特异性转化为胰凝乳蛋白酶的特异性已经显示需要在蛋白质的多个部分中交换氨基酸，包括不直接接触底物的两个表面环。晶体学分析的两个突变体胰蛋白酶具有糜蛋白酶样特异性，现在表明，这些远端表面环改变功能，直接确定的主要结合位点的结构。同源底物的有效酰化与保守Gly216残基的独特骨架构象相关。该氨基酸位于特异性口袋的表面上，并与底物的非特异性部分形成两个主链氢键。相比之下，通过用Trp取代远端Tyr172残基来改善底物结合亲和力效应源自口袋的最底部的结构重排。总之，动力学和晶体学数据强烈表明，Asp189和Gly216必须被认为是胰蛋白酶底物特异性的主要决定因素。
Converting the specificity of trypsin to that of chymotrypsin has been shown to require the exchange of amino acids in multiple portions of the protein, including two surface loops which do not directly contact the substrate. Crystallographic analysis of two mutant trypsins possessing chymotrypsin-like specificity now reveals that these distal surface loops alter function by directly determining the structure of the primary binding site. Efficient acylation of cognate substrates correlates with a distinct backbone conformation of the conserved Gly216 residue. This amino acid is located on the surface of the specificity pocket and forms two main-chain hydrogen bonds with a nonspecific portion of substrate. By contrast, the improvement in substrate binding affinity effect by the substitution of the distal Tyr172 residue with Trp derives from structural rearrangements at the extreme base of the pocket. Together, the kinetic and crystallographic data strongly suggest that both Asp189 and Gly216 must be considered as primary determinants of substrate specificity in trypsin.