Jering H, Tschesche H
Eur J Biochem. 1976 Jan 15;61(2):443-52. doi: 10.1111/j.1432-1033.1976.tb10038.x.
The derivative of the trypsin-kallikrein inhibitor (Kunitz), TKI+, was prepared with the reactive-site peptide bond Lys-15-Ala-16 hydrolyzed. This was achieved by selective borohydride reduction of the Cys-14-Cys-38 disulfide bond, followed by tryptic cleavage of the reactive-site peptide bond, air reoxidation of the half-cystine residues, and purification by ion-exchange chromatography. The derivative corresponds to the hypothetical 'modified' inhibitor TKI+, which so far could not be obtained from virgin inhibitor by a direct modification reaction (partial proteolysis). The derivative isolated was homogeneous as revealed by amino acid analysis, disc electrophoresis, inactivation by carboxypeptidase B, and inactivation by sodium borohydride reduction. The inhibitory activity of the sodium-borohydride-reduced inhibitor was fully recovered after air reoxidation. The site of cleavage in the inhibitor was confirmed by performic acid oxidation and subsequent isolation of the two corresponding peptides containing residues 1-15 and 16-58 of the entire polypeptide chain. From several aminopeptidases tested only aminopeptidase K rapidly cleaved Ala-16 and Arg-17 from the modified inhibitor and at a reduced rate Ile-18. Des-(Ala16,Arg17)-inhibitor and des-Ala16-inhibitor are both lacking a strong inhibitory activity against bovine trypsin. This indicates a decrease in the association constant by factor of at least 10(8)-10(10). The reactive-site-modified inhibitor is not subject to further enzymic breakdown and therefore is a permanent inhibitor of trypsin. However, the modified inhibitor forms the inactive complex much slower than virgin inhibitor. In the modified inhibitor the hydrolyzed peptide bone was resynthesized to yield virgin inhibitor by forming the complex with trypsin and subjecting the complex to kinetic control dissociation. This proves that the bond Lys-15--Ala-16 is at the reactive site of this inhibitor. Preparation of a modified and still active inhibitor (Kunitz) is in agreement with the general model proposed for the interaction of proteinase-inhibitor--proteinase interactions. This presents new evidence that this model is generally applicable.
制备了胰蛋白酶 - 激肽释放酶抑制剂（库尼兹型，Kunitz）的衍生物TKI +，其活性部位的肽键Lys-15-Ala-16已水解。这是通过对Cys-14-Cys-38二硫键进行选择性硼氢化钠还原，随后对活性部位的肽键进行胰蛋白酶裂解，半胱氨酸残基进行空气再氧化，并通过离子交换色谱法纯化来实现的。该衍生物对应于假设的“修饰”抑制剂TKI +，到目前为止，还无法通过直接修饰反应（部分蛋白酶解）从原始抑制剂中获得。通过氨基酸分析、圆盘电泳、羧肽酶B失活以及硼氢化钠还原失活表明，分离得到的衍生物是均一的。空气再氧化后，硼氢化钠还原抑制剂的抑制活性完全恢复。通过过甲酸氧化并随后分离出包含整个多肽链1-15和16-58位残基的两种相应肽段，证实了抑制剂中的裂解位点。在测试的几种氨肽酶中，只有氨肽酶K能快速从修饰抑制剂上裂解下Ala-16和Arg-17，对Ile-18的裂解速率较低。去（Ala16，Arg17） - 抑制剂和去Ala16 - 抑制剂对牛胰蛋白酶均缺乏强抑制活性。这表明结合常数至少降低了10（8） - 10（10）倍。活性部位修饰的抑制剂不会进一步发生酶解，因此是胰蛋白酶的永久性抑制剂。然而，修饰抑制剂形成无活性复合物的速度比原始抑制剂慢得多。在修饰抑制剂中，通过与胰蛋白酶形成复合物并使复合物进行动力学控制解离，将水解的肽键重新合成以产生原始抑制剂。这证明Lys-15 - Ala-16键位于该抑制剂的活性部位。制备修饰且仍具活性的抑制剂（库尼兹型）与所提出的蛋白酶 - 抑制剂 - 蛋白酶相互作用的一般模型一致。这提供了新的证据表明该模型普遍适用。
