Kunitz型丝氨酸蛋白酶抑制剂结构与功能研究

Research on the Structure-function Relationship of Kunitz-type Serine Protease Inhibitors

蛋白酶抑制剂在酶学及蛋白质的结构与功能关系研究中有重要意义,Kunitz型丝氨酸蛋白酶抑制剂是其中最重要的,也是研究最广泛的蛋白酶抑制剂之一.该类蛋白酶抑制剂三维结构高度保守:由一个明显的疏水核心、三对高度保守的二硫键桥、三链β 折叠和一个N端310螺旋及一个C端α 螺旋组成.3对二硫键对分子空间结构的稳定起着非常重要的作用.这一类型抑制剂有5个主要的活性位点:P1、P1′、P3、P3′、P4,它们都位于一个溶剂暴露的环上.P1位点是抑制作用的关键活性位点,抑制剂的专一性由P1位点氨基酸残基的性质决定;P1′位点氨基酸残基的侧链大小对抑制剂 酶的结合常数有很大影响,用大的侧链残基取代会导致结合常数降低;P4位点残基被取代经常产生负效应,会导致活性区域环的构象发生很大改变,从而影响酶与抑制剂的结合.

Proteinase inhibitors play an important role in the research of enzyme studies and the structure function relationship of proteins.The Kunitz family of serine protease inhibitors is one of the most important and extensively studied protease inhibitor families.The structure of this type of protease inhibitors are high conserved.They have a clear hydrophobic core,three disulfide bonds,threestranded βsheet and two short helices Nterminal 310 helix and a Cterminal αhelix.Three disulfide bridges contribute to the overall stability of this class of proteins.They contain a solvent exposed loop of similar conformation which consists of five main active sites:P1,P1′,P3,P3′,P4.A large part of the enzymeinhibitor contacts is made just by the P1 residue,which penetrates deeply into the S1 specificity binding pocket of the protease.The amino acid types at P1 site likely play an important role in causing different inhibitory activities.Mutations of the wild type Ala16(P1′) to larger side chains always caused a drop of the association constant.The substitutions at the P4 site always caused negative effects on the association constant with all the studied enzymes.All P4 mutants showed a very large decrease of the denaturation temperature,suggesting that substitution of the wild type Gly12 residue leads to a significant change in the binding loop conformation.