Synthesis and characterization of a pancreatic trypsin inhibitor homologue and a model inhibitor.
Synthesis and characterization of a pancreatic trypsin inhibitor homologue and a model inhibitor.
复制标题
胰腺胰蛋白酶抑制剂同系物和模型抑制剂的合成和表征。
作者：
N. Tan;E. Kaiser
在酶活性位点附近氨基酸组成变化的蛋白蛋白酶抑制剂同源物的合成和表征应该有助于理解酶活性抑制发生的机制。合成了一种将碱性胰蛋白酶抑制剂（Kunitz） （BPTI）的反应位点残基Ala-16替换为苯丙氨酸（Phe）的同源抑制剂，研究了这种替换对酶抑制剂复合物解离常数的影响。Ala-16被Phe取代导致胰蛋白酶- bpti复合物的K1值急剧增加，而凝乳胰蛋白酶- bpti复合物的K1值基本保持不变。这不能简单地用空间拥挤的增加来解释。Phe的替换可能会导致抑制剂活性位点的局部构象发生微小变化，从而导致非常紧密的胰蛋白酶- bpti复合物的稳定性大大降低。这种构象变化显然可以在结合较不紧密的凝乳胰蛋白酶- bpti复合体中耐受。在已知BPTI结构的基础上，合成一个含一个二硫键的环七肽作为模型抑制剂，以确定是否可以设计一个更小的肽作为胰蛋白酶的高效抑制剂。该七肽包含了参与蛋白酶抑制剂与胰蛋白酶相互作用的BPTI的所有氨基酸残基，其与胰蛋白酶的结合比天然BPTI弱3 × 10(7)倍。因此，即使只有一小部分抑制剂分子直接进入与酶的相互作用，分子中保持抑制剂结构刚性的其余部分对于强相互作用是必不可少的。
The synthesis and characterization of protein proteinase inhibitor homologues with variations in the amino acid composition in the vicinity of the reactive site should aid the understanding of the mechanism by which inhibition of enzymatic activity occurs. A homologue inhibitor in which the reactive-site residue Ala-16 of basic pancreatic trypsin inhibitor (Kunitz) (BPTI) is replaced by Phe has been synthesized to study the effect of this replacement on the dissociation constants of the enzyme-inhibitor complexes. The replacement of Ala-16 by Phe causes a dramatic increase in the K1 value of the trypsin-BPTI complex while that of the chymotrypsin-BPTI complex remains essentially the same. This cannot be explained simply in terms of increased steric crowding. The Phe replacement probably causes a small change in the local conformation of the reactive site of the inhibitor which leads to a large decrease in the stability of the very tight trypsin-BPTI complex. This conformation change apparently can be tolerated in the less tightly bound chymotrypsin-BPTI complex. On the basis of the known structure of BPTI, a cyclic heptadecapeptide containing one disulfide bond was synthesized as a model inhibitor in order to determine if a smaller peptide can be designed to act as a highly efficient inhibitor for trypsin. This heptadecapeptide which contains all of the amino acid residues of BPTI taking part in the interaction of the proteinase inhibitor with trypsin binds 3 X 10(7) time more weakly to the enzyme than native BPTI does. It thus appears that even though only a small part of the inhibitor molecule enters directly into interaction with the enzyme, the remaining portions of the molecule which hold the structure of the inhibitor rigid are essential for the strong interaction.