Escherichia coli prlC gene encodes a trypsin-like proteinase regulating the cell cycle
Escherichia coli prlC gene encodes a trypsin-like proteinase regulating the cell cycle
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DOI：
10.1093/oxfordjournals.jbchem.a022216
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发表时间：
1998-11-01
影响因子：
2.7
通讯作者：
Muramatu, M
中科院分区：
文献类型：
--
作者：
Jiang, XY;Zhang, MY;Muramatu, M
蛋白酶TTL先前被描述为表现出胰蛋白酶样蛋白酶活性，该活性暂时出现在DNA合成之前，在大肠杆菌通过磷酸盐饥饿同步的大肠杆菌的细胞周期中，与DNA复制的启动密切相关[Kato，M.，M.，Irisawa ，T.，Ohtani，RI。和Muramatu，M。（1992）Fur。 J. Biochem。 210，1007-1014]。我们从大肠杆菌C600中纯化了蛋白酶，发现其氨基末端的15个氨基酸残基与大肠杆菌PRLC基因的乘积寡肽A（OPDA）相同。纯化的蛋白酶的分子质量约为67 kDa，与寡肽酶A的分子质量也相同。为了进一步阐明蛋白酶在和寡肽酶A中的关系，我们组装了表达载体以指导大肠杆菌寡肽酶A的合成。重组酶（RPRLC）和纯化的蛋白酶均可用胰蛋白酶底物和苯甲酰氧甲苄醛酶Ala-Ala-Ala-Leu p-Nitroanilide（Z-AALPNA）的形式水解胰蛋白酶底物，描述为各种效应效应的合成底物。 RPRLC上的蛋白酶抑制剂也非常相似蛋白酶in。胰蛋白酶抑制剂4-瓜尼迪诺苯甲酸4-丁基苯基酯和抗蛋白质强烈抑制重组酶的胰蛋白酶样蛋白酶活性，但对其Z-Aalpna水解活性没有影响。大大增强了OPDA活性的钴离子略微抑制了重组酶的胰蛋白酶样活性。这些结果强烈表明，蛋白酶由大肠杆菌PRLC基因编码，是一种具有两个独立活性位点的多功能蛋白酶。
Proteinase Ttl has previously been described as displaying a trypsin-like proteinase activity that momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication [Kato, M., Irisawa, T., Ohtani, RI., and Muramatu, M. (1992) fur. J. Biochem. 210, 1007-1014]. We purified the proteinase In from E. coli C600 and found that the 15 amino acid residues of its amino-terminal were identical with those of oligopeptidase A (OpdA), the product of the E. coli prlC gene. The purified proteinase had a molecular mass of approximately 67 kDa, which was also the same as that of oligopeptidase A. To further elucidate the relationship between proteinase In and oligopeptidase A, we assembled an expression vector to direct the synthesis of E. coli oligopeptidase A. The protein was expressed at a high level in E. coli BL21(DE3) and was produced mostly in the soluble, active form, Both the recombinant enzyme (rPrlC) and the purified proteinase In could hydrolyze trypsin substrates for proteinase In as well as benzyloxycarbonyl Ala-Ala-Leu p-nitroanilide (Z-AALpNA), described as a synthetic substrate for oligopeptidase A. The effects of various protease inhibitors on rPrlC were also very similar to those on proteinase In. The trypsin inhibitors 4-guanidino benzoic acid 4-tert-butylphenyl ester and antipain strongly inhibited the trypsin-like proteinase activity of the recombinant enzyme, but had no effect on its Z-AALpNA hydrolyzing activity. Cobalt ion, which greatly enhanced the OpdA activity, slightly inhibited the trypsin-like activity of the recombinant enzyme. These results strongly suggest that proteinase In is encoded by the E. coli prlC gene and is a multi-functional proteinase with two separate active sites.