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 t he 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 abo lished the enzyme activity, which was confirmed by replacing a 600 bp fragment containing the mutation with the corresponding wild-type sequ ence. The same tryptophen residue was then substituted with an alanine in both enzymes by site-directed mutagenesis to reveal a possible rel ationship between their active sites. The purified alpha-N-acetylgalac tosaminidase mutant demonstrated a specific activity of 2.8 x 10(-2) U /mg and a V-max/K-m of 4.3 x 10(-2), which were both more than a thous andfold lower than corresponding values for the wild-type enzyme. Furt hermore, the mutant failed to bind to an affinity resin, suggesting th e involvement of Trp-16 in substrate-binding. In addition, the purifie d alpha-galactosidase mutant resulted in more than a 10(4)-fold decrea se in specific activity. Thus our data suggest that Trp-16 in both alp ha-galactosidase and alpha-N-acetylgalactosaminidase is critical for e nzymatic activity, which in turn supports the hypothesis that these tw o enzymes may share a catalytic mechanism involving similar residues i n their active sites.