Identification of tyrosine 108 in coffee bean alpha-galactosidase as an essential residue for the enzyme activity.
The cDNA for coffee bean alpha-galactosidase (alpha-Gal) has been cloned and expressed in a baculovirus expression system. An early study of coconut alpha-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 alpha-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 Vmax/Km value for the mutant Y108F was 0.027, which was over a 1000-fold lower than that of wild-type alpha-Gal. Our data suggest that Tyr-108 is critical for the enzymatic activity of alpha-Gal.
Zhu
A
A
Lindsley F. Kimball Research Institute, New York Blood Center, NY 10021.
Wang
Z K
ZK
Goldstein
J
J
D016367:Baculoviridae; D001483:Base Sequence; D002384:Catalysis; D003069:Coffee / Q000201:enzymology*; D018076:DNA, Complementary / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D014443:Tyrosine / Q000032:analysis*; D000519:alpha-Galactosidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D003069:Coffee; D018076:DNA, Complementary; D014443:Tyrosine; D000519:alpha-Galactosidase