Autocatalytic processing of the streptococcal cysteine protease zymogen -: Processing mechanism and characterization of the autoproteolytic cleavage sites
Autocatalytic processing of the streptococcal cysteine protease zymogen -: Processing mechanism and characterization of the autoproteolytic cleavage sites
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DOI：
10.1046/j.1432-1327.1999.00473.x
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发表时间：
1999-07-01
期刊：
影响因子：
--
通讯作者：
Ziomek, E
中科院分区：
文献类型：
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作者：
Doran, JD;Nomizu, M;Ziomek, E
使用来自化脓性链球菌菌株 B220 的纯化蛋白，在体外研究了链球菌半胱氨酸蛋白酶酶原 (proSCP) 向活性链球菌半胱氨酸蛋白酶 (SCP) 的自催化过程。研究发现，酶原的自催化成熟是通过至少六种中间体的连续出现进行的，其中五种中间体通过 N 端测序和 MS 的组合进行了表征。中间体被鉴定为 Lys26、Asn41、Lys101、Ala112 和 Lys118 之后裂解的结果。 proSCP 加工的时间过程研究给出了 S 形活性曲线，并表明 proSCP 主要通过分子间加工机制催化其自身的转化。当用天然的酶促活性 SCP 处理非活性 Cys192Ser proSCP 时，观察到中间体的类似顺序出现，从而证明成熟只能通过双分子机制进行。结果表明，固定在琼脂糖树脂上的 proSCP（而非成熟的 SCP）能够从柱中释放自身，这表明酶原也能够进行分子内加工。为了测试加工位点的氨基酸序列是否可用于开发新的特定底物，基于来自自动加工裂解位点的所有五个特征氨基酸序列合成了3-氨基苯甲酸八肽衍生物并测试了活性。 3-氨基苯甲酸衍生物的k(cat)/K-M值范围为1200至7700.M-1.s(-1)，使其成为SCP非常好的内肽酶底物。
The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N-terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3-amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have k(cat)/K-M values ranging from 1200 to 7700.M-1.s(-1), making them very good endopeptidase substrates for SCP.