Taguchi S, Suzuki M, Kojima S, Miura K, Momose H
Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan.
J Bacteriol. 1995 Nov;177(22):6638-43. doi: 10.1128/jb.177.22.6638-6643.1995.
Previously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter. The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20. From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i.e., a serine protease with broad substrate specificity. For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80 degrees C by the addition of 10 mM calcium ion. The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10(-10) M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI. The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-P20. This reactive site is the same that toward an exogenous target enzyme, subtilisin BPN'.
此前，我们从一株不产生链霉菌枯草杆菌蛋白酶抑制剂（SSI）的突变菌株中分离出一种链霉菌枯草杆菌蛋白酶抑制剂（SSI）的内源性靶酶候选物，命名为SAM-P20（S. 田口、A. 小田、Y. 渡边和H. 桃濑，《应用与环境微生物学》61:180 - 186，1995年）。在本研究中，为了研究这种蛋白酶的详细酶学性质，利用SSI基因启动子在天蓝色链霉菌中建立了重组SAM-P20的过量表达系统。通过盐析和两次连续的离子交换色谱法对重组SAM-P20进行纯化，使其在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中呈现出单一的条带。部分肽图谱分析和氨基酸组成分析表明，重组SAM-P20与天然SAM-P20相同。根据底物特异性和抑制剂敏感性的结果，SAM-P20可归类为具有精氨酸可切割活性的类胰凝乳蛋白酶，即具有广泛底物特异性的丝氨酸蛋白酶。对于蛋白水解活性，最适pH为10.0，通过添加10 mM钙离子，最适温度从50℃转变为80℃。SSI二聚体蛋白对SAM-P20活性具有强烈的化学计量抑制作用，这两种蛋白的亚基摩尔比约为1，估计SSI对SAM-P20的抑制常数为8.0×10⁻¹⁰ M。通过分析凝胶过滤监测SAM-P20和SSI的复合物形成，除了一个SAM-P20分子与一个SSI二聚体分子结合的复合物外，还检测到由两个SAM-P20分子和一个SSI二聚体分子组成的复合物。通过对SSI与SAM-P20复合物酸化后产生的修饰形式的SSI进行序列分析，确定了SSI对SAM-P20的反应位点为Met-73-Val-74。该反应位点与对外源靶酶枯草杆菌蛋白酶BPN'的反应位点相同。
