| 摘要: |
| ［摘要］ 目的 探讨组织蛋白酶E第284位苏氨酸残基在酶活性中的作用。方法 通过基因点突变，将大鼠组织蛋白酶E内羧基端的活性部位基序中第284位苏氨酸残基用丝氨酸进行替代，构建变异体T284S。结果 当将野生型和变异型的大鼠组织蛋白酶E基因在HEK 293T细胞中进行异源表达时，在细胞培养基和细胞提取物中都发现有野生型和变异型的大鼠组织蛋白酶E的酶原蛋白表达。突变体T284S仍具有自我转化为成熟型酶的能力，尽管它们的酶活性只是野生型的40%，变异体的Km值与野生型的相似，但它的Kcat值却比野生型显著下降。变异体和野生型酶都被胃酶抑素和蛔虫胃蛋白酶抑制剂所抑制，Ki值几乎相同。变异体在各种酸碱度下的圆二色光谱与野生型基本相同。结论 （1）第284位苏氨酸残基对酶的催化活性确实非常重要。变异体T284S酶催化活性的下降是由于多出来的甲基使活性部位氢键的分布发生改变，从而使酶与底物的作用模式发生改变。（2）BACE-1不能被胃酶抑素抑制，可能与羧基端结构域的活性部位基序差异无关，而与BACE1蛋白结构中的其它部分有关。 |
| 关键词: 活性部位基序 天门冬氨酸蛋白酶 组织蛋白酶E 变异体解析 |
| DOI：10.3969/j.issn.1674-3806.2009.07.01 |
| 分类号:R 963 |
| 基金项目:广东省科技计划项目资助(项目编号:2008B060600055) |
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| The role of Thr284 in the catalysis of Cathepsin E |
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LIU Jian
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School of Life Science and Biopharmacology, Guangdong pharmacological College, Guangzhou 510006, China
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| Abstract: |
| ［Abstract］ Objective To investigate the role of Thr284 in the catalysis of Cathepsin E.Methods Replace Thr284 with a serine to produce a T284S mutant by site-directed mutagenesis.Results Both wild type and mutant are expressed and present in the HEK 293T cell culture media and cell lysate. Mutant is still able to auto activate, although its activity is only 40% of that of wild type. The Km value of mutant is similar to that of wild type, but its Kcat value is smaller than that of wild type. Both mutant and wild type are inhibited by pepstatin and Ascaris pepsin inhibitor with similar Kivalues. The CD spectra of mutant and wild type is similar.Conclusion (1)Thr284 is very important to the catalytic activity. The mutant causes change of hydrogen bonds distribution and thus lowers the hydrolysis efficiency. (2)Other protein structural features of BACE-1 than its carboxyl motif may contribute to its insensitivity to pepstatin. |
| Key words: Active site motif Aspartic proteinase Cathepsin E Mutant analysis |
