Identification of tyrosine 108 in coffee bean α-galactosidase as an essential residue for the enzyme activity

Abstract

The cDNA for coffee bean α-galactosidase (α-Gal) has been cloned and expressed in a baculovirus expression system. An early study of coconut α-Gal by chemical modification suggested that one tyrosine residue is at or near the active site. In order to identify such a critical residue, we replaced two tyrosine residues (positions 108 and 158) with phenylalanine by site-directed mutagenesis. The mutated DNA strands, as well as the wild-type ones, were subcloned into pVL vector and transformed into Sf9 insect cells for intracellular expression. The replacement of Tyr-158 with phenylalanine resulted in a mutant α-Gal (Y158F) which retained approx. 88% of the activity of wild-type enzyme. However, the substitution of Tyr-108 by phenylalanine (Y108F) almost abolished the enzymatic activity (1.8% of wild-type activity). The V max K m value for the mutant Y108F was 0.027, which was over a 1000-fold lower than that of wild-type α-Gal. Our data suggest that Tyr-108 is critical for the enzymatic activity of α-Gal.