Effect of Urea on Activity and Conformation of a Glycoprotein |
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Authors: | WEI Xiang WANG Xiaoyun ZHOU Bo ZHOU Haimeng |
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Affiliation: | Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China; College of Life Science, Shandong Agricultural University, Taian 271018, China |
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Abstract: | The changes of the activity and conformation of Aspergillus niger phytase in urea were detected by far-ultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays. The results show that no enzyme activity can be detected after phytase is incubated for 10 h in 3.0 mol/L urea, even though at this urea concentration, less than 20% of the tertiary and secondary structures in the native enzyme changed. The inactivation reaction kinetics is found to be a monophasic first-order reaction, but the unfolding is a biphasic process consisting of two first-order reactions. The inactivation rates of the free enzyme and the substrate-enzyme complex are much faster than the conformational changes during urea denaturation. All of the results indicate that, as a glycoprotein, phytase's activity is strongly dependent on its conformational integrity. The phytase active sites seem to be located in a limited region in the molecule and display more conformational fragility and flexibility to denaturants than enzyme molecular structure as a whole. |
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Keywords: | phytase urea denaturation inactivation unfolding |
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