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.
Zhu
A
A
Lindsley F. Kimball Research Institute, New York Blood Center, NY 10021, USA.
Monahan
C
C
Wang
Z K
ZK
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003069:Coffee / Q000201:enzymology; D006596:Hexosaminidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014364:Tryptophan / Q000502:physiology*; D000519:alpha-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D048809:alpha-N-Acetylgalactosaminidase
D003069:Coffee; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D006596:Hexosaminidases; D000519:alpha-Galactosidase; D048809:alpha-N-Acetylgalactosaminidase