CONVERTING TRYPSIN TO CHYMOTRYPSIN - THE ROLE OF SURFACE LOOPS
CONVERTING TRYPSIN TO CHYMOTRYPSIN - THE ROLE OF SURFACE LOOPS
复制标题
DOI：
10.1126/science.1546324
复制
发表时间：
1992-03-06
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：
HEDSTROM, L;SZILAGYI, L;RUTTER, WJ
胰蛋白酶（Tr）和胰凝乳蛋白酶（Ch）具有相似的三级结构，但Tr在精氨酸和赖氨酸残基上切割肽，而Ch更喜欢大的疏水残基。虽然用类似的Ch残基取代Tr的S1结合位点足以转移Ch对酯水解的特异性，但不会转移酰胺水解的特异性。通过将Ch表面环185 - 188和221 - 225交换为类似的Tr环，进一步修饰结合袋改变，胰蛋白酶转化为Ch样蛋白酶。这些环不是S1结合位点或延伸底物结合位点的结构成分。该突变酶的催化速率与Ch相当，但其与底物的结合受到损害。与Ch一样，该突变体利用扩展底物结合来加速催化，并且底物识别发生在酰化步骤而不是底物结合过程中。
Trypsin (Tr) and chymotrypsin (Ch) have similar tertiary structures, yet Tr cleaves peptides at arginine and lysine residues and Ch prefers large hydrophobic residues. Although replacement of the S1 binding site of Tr with the analogous residues of Ch is sufficient to transfer Ch specificity for ester hydrolysis, specificity for amide hydrolysis is not transferred. Trypsin is converted to a Ch-like protease when the binding pocket alterations are further modified by exchange of the Ch surface loops 185 through 188 and 221 through 225 for the analogous Tr loops. These loops are not structural components of either the S1 binding site or the extended substrate binding sites. This mutant enzyme is equivalent to Ch in its catalytic rate, but its substrate binding is impaired. Like Ch, this mutant utilizes extended substrate binding to accelerate catalysis, and substrate discrimination occurs during the acylation step rather than in substrate binding.