Role of Ser-65 in the activity of alpha-galactosidase A: characterization of a point mutation (S65T) detected in a patient with Fabry disease.
Fabry disease is a genetic disorder caused by deficient activity of alpha-galactosidase A (alpha-Gal A). Recent gene analysis of a Fabry patient revealed a point mutation (S65T) resulting in a significant decrease of enzyme activity (Chen, C.-H., et al. (1998) Hum. Mutat. 11, 328-330). In order to evaluate the role of Ser-65 in the alpha-Gal A activity and the molecular mechanism of its deficient enzyme activity in mammalian cells, we prepared gene products of S65T, S65A, and E66D mutations of alpha-Gal A by using an expression system with baculovirus/insect cells and characterized the kinetic and physical properties of those purified enzymes. The Km values of mutant enzymes were 3.5 (S65T), 3.4 (S65A), and 2.3 mM (E66D), using 4-methylumbelliferyl alpha-D-galactoside as a substrate, and the Vmax values were 2.7 x 10(6) (S65T), 3.4 x 10(6) (S65A), and 2.5 x 10(6) units/mg (E66D), respectively, which were similar to those of the normal enzyme (Km, 2.3 mM; Vmax, 2.3 x 10(6) units/mg). The in vitro stability of mutant enzymes at neutral pH was significantly reduced (S65T, 4% of normal; S65A, 29%; E66D, 54%). The intracellular alpha-Gal A activities of S65T, S65A, and E66D in COS1 cells transfected with corresponding plasmid DNAs were markedly lower than the normal enzyme activity (9, 26, and 68% of normal, respectively). However, intracellular enzyme activities were enhanced to 34% (S65T), 44% (S65A), and 80% (E66D) of normal, respectively, by cultivation of the cells with 20 microM 1-deoxygalactonojirimycin (a potent inhibitor of alpha-Gal A) for 24 h. These results suggest that Ser-65 is responsible for the stability of alpha-Gal A but not for the enzyme function.
Ishii
S
S
The Usuki Bio Research Center, Oita, Japan. uskishii@fat.coara.or.jp
Suzuki
Y
Y
Fan
J Q
JQ
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000795:Fabry Disease / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D012694:Serine / Q000235:genetics; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D000519:alpha-Galactosidase / Q000235:genetics*
D018076:DNA, Complementary; D012694:Serine; D000519:alpha-Galactosidase