Li Y, Huang Q, Zhang S, Liu S, Chi C, Tang Y
Institute of Physical Chemistry, Peking University, Beijing, China.
J Biochem. 1994 Jul;116(1):18-25. doi: 10.1093/oxfordjournals.jbchem.a124491.
The active fragment with Lys at the reactive site of mung bean trypsin inhibitor (MBILF) is composed of two peptide chains, A1 of 26 residues and A2 of 9 residues linked via two disulfide bonds. In the present study, a peptide of 22 residue comprising the sequence of chain A1 from position 3 to 24 was synthesized by the solid-phase method. This synthetic peptide with six Cys residues contains a reactive site at position Lys11I-Ser12I (I denotes an inhibitor residue). Air oxidation and HPLC purification resulted in two antitrypsin active components, SPC1 and SPC2. Neither SPC1 nor SPC2 can stoichiometrically inhibit trypsin. The Ki values of SPC1 and SPC2 are 1.2 x 10(-7) and 4.0 x 10(-8) M, respectively. The complexes of SPC1 and SPC2 with bovine beta-trypsin (BTRY) were crystallized by ammonium sulphate precipitation at pH 6.4 and 6.0, respectively. The two crystals have the same crystal form with space group P2(1)2(1)2(1) and cell dimension of a = 63.2(2) A, b = 63.5(6) A, and c = 69.8(4) A. The crystal structure of one complex, SPC1-BTRY, was determined and refined at 2.2 A resolution to a final R-value of 19.2%. From the resulting electron density map, 9 residues of SPC1, from position 9I to 17I, were identified clearly and three-dimension atomic model of the 9-residue reactive loop formed by a disulfide bridge, Cys9I-Cys17I, was built. No electron density corresponding to the other 13 residues was observed in the present map.(ABSTRACT TRUNCATED AT 250 WORDS)
绿豆胰蛋白酶抑制剂（MBILF）活性位点带有赖氨酸的活性片段由两条肽链组成，即含26个残基的A1链和含9个残基的A2链，二者通过两条二硫键相连。在本研究中，采用固相法合成了一条含22个残基的肽段，其包含A1链从第3位到第24位的序列。这条含有6个半胱氨酸残基的合成肽在Lys11I - Ser12I位点（I表示抑制剂残基）含有一个活性位点。空气氧化和高效液相色谱纯化得到了两种抗胰蛋白酶活性成分，即SPC1和SPC2。SPC1和SPC2均不能以化学计量方式抑制胰蛋白酶。SPC1和SPC2的抑制常数（Ki）值分别为1.2×10⁻⁷和4.0×10⁻⁸ M。SPC1和SPC2与牛β-胰蛋白酶（BTRY）的复合物分别通过在pH 6.4和6.0条件下硫酸铵沉淀法结晶。这两种晶体具有相同的晶型，空间群为P2(1)2(1)2(1)，晶胞参数为a = 63.2(2) Å，b = 63.5(6) Å，c = 69.8(4) Å。确定了其中一种复合物SPC1 - BTRY的晶体结构，并在2.2 Å分辨率下进行精修，最终R值为19.2%。从所得电子密度图中，清晰鉴定出了SPC1从第9I位到第17I位的9个残基，并构建了由二硫键Cys9I - Cys17I形成的9个残基活性环的三维原子模型。在当前图中未观察到与其他13个残基对应的电子密度。（摘要截短至250字）
