An acidic amino acid in aspartic proteinase active site motif
10.3969/j.issn.1673-8225.2009.28.033
- VernacularTitle:一种天门冬氨酸蛋白酶活性部位基序中酸性氨基酸的作用
- Author:
Jian LIU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(28):5558-5561
- CountryChina
- Language:Chinese
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Abstract:
BACKGROUND: Cathepsin E, an intracellular asparUc proteinase of pepsin family, Is composed of 2 homologous domains, each containing a consensus DTG motif, in which the role of aspartic acid (Asp98) is still unknown. OBJECTIVE: To determine the role of Asp98 in the catalysis of Cathepsin E. DESIGN, TIME AND SETTING: An observation experiment was performed in the Guangdong Pharmacological College between November 2007 and December 2008.MATERIALS: Rat Cathepsin E antibody was prepared according to the references, and the purified with affinity chromatography. PcDNA3.0 was offered from Guangzhou Ganewindows Biotech Ltd. Human embryo kidney 293 cell strain was offered from Stem Cell Bank,Chinese Academy of Sciences.METHODS: The cDNA containing full-length rat Cathepsln E was inserted into pBluescdpt IISK plasmid between Smal and Xhol. Asp98 was replaced with an acidic amino acid termed glutamic acid to produce a D98E mutant by Kunkel gene site-directad mutagenesis and expressed in human embryo renal cells.MAIN OUTCOME MEASURES: Expression of wild type and mutant protein was determined, the wild type and mutant Cathepsin E was purified, the enzyme activity, folding and self-precessing were analyzed. RESULTS: Hetaroganous expression of wild type and mutant rat Cathepsin E were found in human embryo kidney 293 cells, and both were expressed in the cell culture media and cell lysate. Mutant D98E was still able to auto-activate into mature enzyme, although its activity was only 13% of that of wild type. The Km value of mutant was similar to that of wild type, but its K<,cat> value was significantly smaller than that of wild type. Both mutant and wild type were inhibited by pepstatin and Ascaris pepsin inhibitor with similar Ki values. The CD spectra of mutant and wild type were similar.CONCLUSION: The Asp98 plays an important role in catalysis, and it can be partly replaced by glutamic acid. Mutant D98E has a lowered activity since the mutation affects the catalytic efficiency.
