The effect of carbohydrate removal on the properties of human liver alpha-L-fucosidase.
The effect of carbohydrate removal on the properties of the lysosomal enzyme alpha-L-fucosidase has been investigated by comparatively characterizing N-glycanase-treated and mock-treated control fucosidases. N-Glycanase treatment removed approx. 90% of the carbohydrate from purified native human liver fucosidase as determined by carbohydrate assay after gel filtration on Sephadex G-50, and by Western blotting with a lectin-digoxigenin conjugate and densitometric scanning. Removal of carbohydrate from fucosidase does not affect its catalytic activity, its Km value for synthetic substrate, its recognition and rate of hydrolysis of three natural substrates, or its gross conformation as determined by circular dichroism. However, loss of carbohydrate led to significantly decreased activity at acidic pH values (3.1-4.7), a 0.6 pH unit shift to a more neutral optimum and decreased thermostability. The decreased activity at acidic pH values and the more neutral pH optimum of deglycosylated fucosidase suggest that the presence of carbohydrate is physiologically significant in allowing fucosidase to perform its catabolic function more efficiently in the acidic milieu of the lysosome.
Piesecki
S
S
Department of Chemistry, Lehigh University, Bethlehem, PA.
Alhadeff
J A
JA
D000581:Amidohydrolases / Q000737:chemistry; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000302:isolation & purification*; D002942:Circular Dichroism; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011487:Protein Conformation; D005644:alpha-L-Fucosidase / Q000737:chemistry / Q000302:isolation & purification*
D002241:Carbohydrates; D009844:Oligosaccharides; D005644:alpha-L-Fucosidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase