Kojima S, Nishiyama Y, Kumagai I, Miura K
Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo.
J Biochem. 1991 Mar;109(3):377-82. doi: 10.1093/oxfordjournals.jbchem.a123389.
It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)
已表明蛋白酶抑制剂的P1位点（活性位点中心）与同源蛋白酶的特异性相对应，因此通过在P1位点替换单个氨基酸可以改变枯草芽孢杆菌蛋白酶抑制剂（SSI）的特异性。在本文中，为了研究对于具有广泛底物特异性的枯草杆菌蛋白酶BPN'，是否能观察到突变型SSI的抑制活性与蛋白酶底物偏好之间存在类似的相关性，通过盒式诱变和定点诱变构建了反应位点P1（第73位）处SSI的全套突变体，并测定了它们对枯草杆菌蛋白酶BPN'的抑制活性。在P1位点具有极性（丝氨酸、苏氨酸、谷氨酰胺、天冬酰胺）、碱性（赖氨酸、精氨酸）或芳香族氨基酸（酪氨酸、苯丙氨酸、色氨酸、组氨酸），或丙氨酸或亮氨酸的突变型SSI对枯草杆菌蛋白酶显示出与野生型（甲硫氨酸）SSI几乎相同的强抑制活性。然而，在P1位点具有酸性（谷氨酸、天冬氨酸）、β-分支脂肪族氨基酸（缬氨酸、异亮氨酸），或甘氨酸或脯氨酸的SSI变体的抑制活性降低。突变型SSI对枯草杆菌蛋白酶BPN'的抑制常数（Ki）值与枯草杆菌蛋白酶BPN'的底物偏好一致。在突变型SSI的log(1/Ki)与合成底物的log(1/Km)之间观察到线性相关性。这些结果表明SSI的P1位点突变体的抑制活性与枯草杆菌蛋白酶BPN'的底物偏好呈线性相关，并表明抑制剂与蛋白酶的结合模式可能与底物的结合模式相似，就像胰蛋白酶和胰凝乳蛋白酶的情况一样。（摘要截短为250字）
