应用定点突变与分子模建方法研究蛋白酶抑制剂eglin C突变体对蛋白酶furin和kexin的抑制活力

蛋白质前体加工酶参与许多重要蛋白质闪体的加工成熟过程，哺乳动物来源的furin和酵母中的kexin是该家族的重要成员。首先人工合成了编码枯草杆菌蛋白酶抑制剂eglin C的基因片段，组装后在大肠杆菌中得到表达。以定点突变方法在野生型eglin C抑制活性中心的P1、P2和P4位引入碱性氨基酸残基可以将其改造为很强的furin抑制剂（Ki约10^-9mol/L），和kexin抑制剂（Ki约10^-11mol/L）。同时根据枯草杆菌蛋白酶和eglin C复合物的晶体结构，计算机同源模建了前体加工酶与eglin C突变体结构之间的相互作用，并结合实验数据得到以下结果：（1）P1位引入的碱性残基是该抑制剂活力的前提；（2）P4位碱性残基的引入可以极大地提高抑制剂活力约两个数量级；（3）P2 的碱性残基将有效提高抑制剂的活力。然而同时可以破坏抑制剂本身的稳定性。（4）野生型P3位的疏水性残基参与抑制剂活性环附近疏水核心的构成。

The Inhibitory Activities of Recombinant Eglin C Mutants on Kexin and Furin, Using Site-directed Mutagenesis and Molecular Modeling

Mammalian furin and yeast kexin are members of the proprotein convertase family involved in the proteolytic processing of many important precursor proteins. Here the gene coding for the subtilisin inhibitor eglin C was totally synthesized and expressed in E.coli. Substitution of residues at each position P 1, P 2 and P 4 of eglin C with a basic residue using protein engineering could make eglin C a very strong inhibitor for furin ( K i around 10 -9 mol/L), and even more strong for kexin ( K i around 10 -11 mol/ L). Results indicated that: (1) A basic residue Lys or Arg at P 1 site is prerequisite for the inhibitor. (2) The second mutation with basic residue at P 4 site drastically increase the inhibitory activity by two orders of magnitude. (3) A basic residue at P 2 site is favorable for the binding to the enzyme, but unfavorable for the stability of the inhibitor, resulting in a temporary inhibition. (4) A hydrophobic residue is preferential at P 3 site. Based on the known crystal structures of subtilisin and eglin C, the interaction between the enzyme and inhibitor was modeled, and their involved residues were predicted which gave a good explanation to the experimental results.