Role of Ser-65 in the Activity of α-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 α-galactosidase A (α-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 α-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 α-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 α-d-galactoside as a substrate, and the Vmax values were 2.7 × 106 (S65T), 3.4 × 106 (S65A), and 2.5 × 106 units/mg (E66D), respectively, which were similar to those of the normal enzyme (Km, 2.3 mM; Vmax, 2.3 × 106 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 α-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 μM 1-deoxygalactonojirimycin (a potent inhibitor of α-Gal A) for 24 h. These results suggest that Ser-65 is responsible for the stability of α-Gal A but not for the enzyme function.