Kinetic and biophysical characterization of a lysosomal α-l-fucosidase from the fresh water mussel, Lamellidens corrianus

Abstract

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.