Role of the S' subsites in serine protease catalysis. Active-site mapping of rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni.
Role of the S' subsites in serine protease catalysis. Active-site mapping of rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni.
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DOI：
10.1021/bi00180a020
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发表时间：
1994-04
期刊：
影响因子：
2.9
通讯作者：
Volker Schellenberger;Christoph W. Turck;William J. Rutter
中科院分区：
文献类型：
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作者：
Volker Schellenberger;Christoph W. Turck;William J. Rutter
通过测量 100 个五肽亲核试剂的酰基转移反应，研究了来自曼氏血吸虫的四种同源丝氨酸蛋白酶、大鼠胰凝乳蛋白酶、大鼠胰蛋白酶、α-裂解蛋白酶和尾蚴蛋白酶的 S' 亚位点特异性。合成了通用结构H-Xaa-Ala-Ala-Ala-Ala-NH2、H-Ala-Xaa-Ala-Ala-Ala-NH2和H-Ala-Ala-Xaa-Ala-Ala-NH2的肽，其中Xaa是D-Ala、Cit和除Cys之外的所有天然氨基酸。这些亲核试剂的可变残基在酰基转移反应中占据 P'1、P'2 和 P'3 位置。发现 P'1 和 P'2 残基对亲核试剂效率的影响超过 2 个数量级，而 S'3 亚位点在所有四种酶中显示出较低的特异性。我们合成了通用结构 H-aa1-aa2-aa3-Ala-Ala-NH2 的共有肽，其中两个或三个位置被氨基酸占据，在第一系列亲核试剂中表现出最高的特异性。在 P'2 和 P'3 位点具有最佳氨基酸残基的肽在胰凝乳蛋白酶和胰蛋白酶催化的反应中表现出非常高的效率。否则，亲核试剂 P'1 和 P'3 位置上的大特异性侧链由于空间位阻而显示出小于附加结合贡献。胰凝乳蛋白酶催化的酰基转移反应与结构 H-Xaa-Leu-Arg-Ala-Ala-NH2 和 H-Xaa-Ala-Ala-Ala-Ala-NH2 的亲核体的比较揭示了这两个系列的显着不同的 P'1 特异性，这证实了大 P'1 和 P'3 残基之间的空间位阻。（摘要在 250 处截断）话）
The S' subsite specificity of four homologous serine proteases, rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni, was studied by measuring acyl-transfer reactions to 100 pentapeptide nucleophiles. Peptides of the general structures H-Xaa-Ala-Ala-Ala-Ala-NH2, H-Ala-Xaa-Ala-Ala-Ala-NH2, and H-Ala-Ala-Xaa-Ala-Ala-NH2 were synthesized, where Xaa is D-Ala, Cit, and all natural amino acids except Cys. The variable residues of these nucleophiles occupy the P'1, P'2, and P'3 positions in acyl-transfer reactions. The P'1 and P'2 residues were found to influence the efficiency of the nucleophiles by more than 2 orders of magnitude, whereas the S'3 subsite shows a lower specificity in all four enzymes. We synthesized consensus peptides of the general structure H-aa1-aa2-aa3-Ala-Ala-NH2, in which two or three positions were occupied by amino acids that showed the highest specificity in the first series of nucleophiles. Peptides with optimal amino acid residues in the P'2 and P'3 positions show a very high efficiency in chymotrypsin- and trypsin-catalyzed reactions. Otherwise, large specific side chains in the P'1 and P'3 positions of the nucleophiles show less than additive binding contributions due to steric hindrance. Comparison of chymotrypsin-catalyzed acyl-transfer reactions to nucleophiles of the structures H-Xaa-Leu-Arg-Ala-Ala-NH2 and H-Xaa-Ala-Ala-Ala-Ala-NH2 reveals a significantly different P'1 specificity for both series which confirms steric hindrance between large P'1 and P'3 residues.(ABSTRACT TRUNCATED AT 250 WORDS)