Kinetic and biophysical characterization of a lysosomal α-l-fucosidase from the fresh water mussel, Lamellidens corrianus
| Autor: | Ashapogu Venugopal, Nadimpalli Siva Kumar, C. Sudheer Kumar, Musti J. Swamy |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Guanidinium chloride Biochemistry 03 medical and health sciences chemistry.chemical_compound Hydrolysis Guanidinium thiocyanate Peptide mass fingerprinting Structural Biology Enzyme Stability Animals Enzyme kinetics Molecular Biology Protein secondary structure Protein Unfolding alpha-L-Fucosidase chemistry.chemical_classification 030102 biochemistry & molecular biology Molecular mass Temperature General Medicine Hydrogen-Ion Concentration Bivalvia Molecular Weight Kinetics 030104 developmental biology Enzyme chemistry Lysosomes |
| Zdroj: | International Journal of Biological Macromolecules. 104:432-441 |
| ISSN: | 0141-8130 |
| DOI: | 10.1016/j.ijbiomac.2017.06.050 |
| Popis: | Kinetic and biophysical studies have been carried out on a lysosomal α-l-fucosidase purified from the fresh water mussel, Lamellidens corrianus. The enzyme migrates as a single band in SDS-PAGE as well as native PAGE corresponding to a Mr of 56kDa. Mass spectrometric analysis yielded a molecular mass of 56175.1Da for the enzyme, and peptide mass fingerprinting studies showed that it shares sequence homology with other fucosidases. Zymogram analysis showed that the α-l-fucosidase hydrolyzed 4-methyl umbelliferyl α-l-fucopyranoside. The pH and temperature optima of the enzyme were found to be 5.0-6.0 and 60°C, respectively. The KM, Vmax and kcat values of the enzyme estimated with p-nitrophenyl fucopyranoside are 0.85mM, 27.20 mU/mL and 1.01s-1, respectively. The inhibition constant (Ki) of the enzyme towards l-Fucose is 1.09mM. CD spectral analysis has shown that the protein contains predominantly β-sheets in its secondary structure. Chemical unfolding studies indicate that α-l-fucosidase unfolds in a broad sigmoidal, cooperative unfolding transition, centered at ∼2.2M for both guanidinium chloride and guanidinium thiocyanate. The present results obtained with the L. corrianus α-l-fucosidase are expected to provide further insights into the various biological processes associated with fucosidases and help in exploiting this enzyme in therapeutic applications. |
| Databáze: | OpenAIRE |
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