Wells J A, Fairbrother W J, Otlewski J, Laskowski M, Burnier J
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080.
Proc Natl Acad Sci U S A. 1994 May 10;91(10):4110-4. doi: 10.1073/pnas.91.10.4110.
Recently, a 29-residue cyclic peptide was synthesized (TrPepz) that was reported to possess nearly the same catalytic activity and specificity as the pancreatic serine protease, trypsin, for hydrolysis of a small ester substrate, N-tosyl-L-arginine methyl ester (TAME), and small and large peptides [Atassi, M. Z. & Manshouri, T. (1993) Proc. Natl. Acad Sci. USA 90, 8282-8286]. To study these results we have resynthesized TrPepz and a related cyclic peptide reported to possess some trypsin-like activity. The authenticity of each peptide was confirmed by mass spectrometry, peptide sequencing, compositional analysis, and 1H NMR spectroscopy. However, neither peptide exhibited any detectable esterase activity or amidase activity under a variety of conditions tested. Molecular modeling studies indicated it was possible for TrPepz to be nearly superimposed upon the active site of trypsin. However, NMR experiments showed the structure of the cyclic peptide to be disordered. Thus, we were unable to confirm the results of Atassi and Manshouri. Our results are consistent with the view that serine protease activity depends not only on the presence of catalytic groups but also on their precise and stable alignment.
最近，合成了一种由29个氨基酸残基组成的环肽（TrPepz），据报道，它对小酯底物N-甲苯磺酰-L-精氨酸甲酯（TAME）以及小肽和大肽的水解反应具有与胰腺丝氨酸蛋白酶胰蛋白酶几乎相同的催化活性和特异性[阿塔西，M. Z. & 曼苏里，T.（1993年）《美国国家科学院院刊》90，8282 - 8286]。为了研究这些结果，我们重新合成了TrPepz以及一种据报道具有某些胰蛋白酶样活性的相关环肽。通过质谱分析、肽测序、成分分析和1H核磁共振光谱对每种肽的真实性进行了确认。然而，在各种测试条件下，这两种肽均未表现出任何可检测到的酯酶活性或酰胺酶活性。分子模拟研究表明，TrPepz有可能几乎与胰蛋白酶的活性位点重叠。然而，核磁共振实验表明该环肽的结构是无序的。因此，我们无法证实阿塔西和曼苏里的结果。我们的结果与以下观点一致，即丝氨酸蛋白酶活性不仅取决于催化基团的存在，还取决于它们精确且稳定的排列。
