Trp-16 is essential for the activity of alpha-galactosidase and alpha-N-acetylgalactosaminidase

By expressing site-directed mutants in the methylotrophic yeast strain Pichia pastoris, the role of a tryptophan residue at position 16 in the activity of alpha-galactosidase and alpha-N-acetylgalactosaminidase, two closely related exoglycosidases, was studied. A substitution of Trp-16 with an arginine residue in alpha-N-acetylgalactosaminidase abolished the enzyme activity, which was confirmed by replacing a 600 bp fragment containing the mutation with the corresponding wild-type sequence. The same tryptophan residue was then substituted with an alanine in both enzymes by site-directed mutagenesis to reveal a possible relationship between their active sites. The purified alpha-N-acetylgalactosaminidase mutant demonstrated a specific activity of 2.8 x 10(-2) U/mg and a Vmax/K(m) of 4.3 x 10(-2), which were both more than a thousandfold lower than corresponding values for the wild-type enzyme. Furthermore, the mutant failed to bind to an affinity resin, suggesting the involvement of Trp-16 in substrate-binding. In addition, the purified alpha-galactosidase mutant resulted in more than a 10(4)-fold decrease in specific activity. Thus our data suggest that Trp-16 in both alpha-galactosidase and alpha-N-acetylgalactosaminidase is critical for enzymatic activity, which in turn supports the hypothesis that these two enzymes may share a catalytic mechanism involving similar residues in their active sites.