Substrate specificity and other properties of α-L-fucosidase from human serum

α-L-Fucosidase from human serum was purified 130,452-fold with a 43% yield. The final specific activity was 10,658 nmol of fucose released/min/mg of protein using p-nitrophenyl-α-L-fucoside as substrate. In natural glycoconjugates, α-L-fucose occurs linked to position 2 of galactose or to position 3, 4, or 6 of N-acetyl-glucosamine. Substrate specificity studies of the purified serum enzyme with synthetic fucosides revealed that certain compounds with α-L-fucose linked to position 2 of galactose were effectively hydrolyzed. Representative of these are the disaccharide Fuc α1→2 Gal and its aryl derivative Fuc α1→2 Gal β1→OC₆H₄NO₂(p) which were hydrolyzed at rates of 741 and 3329 nmol/min/mg of protein, respectively. In comparison the disaccharides Fuc α1→3 GlcNAc, Fuc α1→4GlcNAc, and Fuc α1→6 GlcNAc were ineffectively hydrolyzed at rates of 83, 74 and 54 nmol/min/mg of protein, respectively. However, the aryl disaccharides, Fuc α1→3GlcNAcβ1→OC₆H₅ and Fuc α1→4GlcNAc β1→OC₆H₅, were cleaved at rates 11- and 53-fold faster than their respective simple disaccharides. In contrast, Fuc α1→6 GlcNAc β1→OC₆H₄NO₂(p) was slowly hydrolyzed (73 nmol/min/mg of protein). Polyacrylamide gel electrophoreses of the purified enzyme revealed a single species of protein. Analytical isoelectric focusing resolved the enzyme into seven bands of protein with pI from 4.52 to 4.96. The ability to hydrolyze the aryl glycosides with α-L-fucose linked to position 2 of galactose or to positions 3 or 4 of N-acetylglucosamine accompanied the single electrophoretic species as well as each isoelectric form. The molecular weight of the enzyme as determined by gel filtration at pH 5.0 was 390,000 and at pH 7.0 was 270,000. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated a single subunit with a molecular weight of 63,000. The enzyme has a pH optimum of 5.0, an activation energy of 12,350 cal, and a temperature coefficient of 1.9.