Goldenberg D P, Berger J M, Laheru D A, Wooden S, Zhang J X
Department of Biology, University of Utah, Salt Lake City 84112.
Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5083-7. doi: 10.1073/pnas.89.11.5083.
In a previous study, a genetic screening procedure was used to identify variants of bovine pancreatic trypsin inhibitor that can fold to an active conformation but that are inactivated much more rapidly than the wild-type protein in the presence of dithiothreitol (DTT). The mechanisms by which 30 of these DTT-sensitive variants are inactivated have now been investigated. Some of the amino acid replacements cause rapid inactivation in the presence of DTT because the three disulfides of the native protein are reduced up to 300-fold faster than for the wild-type protein, leading to complete unfolding. Other substitutions, however, do not greatly increase the rate of complete reduction and unfolding but lead to accumulation of an inactive two-disulfide species. There is a striking correlation between the locations of the DTT-sensitive amino acid replacements in the three-dimensional structure of the protein and the mechanisms by which the variants are inactivated. All of the substitutions that cause rapid unfolding are clustered at one end of the folded protein, in the vicinity of the two disulfides that are reduced most slowly during unfolding of the wild-type protein, while substitutions of the other class are all located at the other end of the protein, near the trypsin binding site. These results indicate that the kinetic stability of native bovine pancreatic trypsin inhibitor and its ability to function as a protease inhibitor are largely influenced by residues in two distinguishable regions of the folded protein.
在之前的一项研究中，采用了一种基因筛选程序来鉴定牛胰蛋白酶抑制剂的变体，这些变体能够折叠成活性构象，但在二硫苏糖醇（DTT）存在的情况下，其失活速度比野生型蛋白快得多。现在已经研究了其中30种对DTT敏感的变体的失活机制。一些氨基酸替换在DTT存在下会导致快速失活，因为天然蛋白的三个二硫键的还原速度比野生型蛋白快300倍，导致完全展开。然而，其他替换并没有显著提高完全还原和展开的速率，但会导致无活性的二硫键物种的积累。在蛋白质三维结构中对DTT敏感的氨基酸替换位置与变体失活机制之间存在显著相关性。所有导致快速展开的替换都聚集在折叠蛋白的一端，靠近野生型蛋白展开过程中还原最慢的两个二硫键，而另一类替换都位于蛋白的另一端，靠近胰蛋白酶结合位点。这些结果表明，天然牛胰蛋白酶抑制剂的动力学稳定性及其作为蛋白酶抑制剂的功能能力在很大程度上受到折叠蛋白两个可区分区域中残基的影响。
