GM2 Gangliosidosis B1 Variant: Biochemical and Molecular Characterization of Hexosaminidase A

The biochemical properties of hexosaminidase A (HexA) and the coding sequence of the α-subunit were examined in a patient of Syrian ancestry with the B1 form of Tay-Sachs disease (TSD). The biochemical characteristics of the variant HexA suggest that both active sites are affected by the mutation(s). Kinetic studies with the β-subunit specific substrate, 4 methylumbelliferyl-β-D-N-acetylglucosamine (MUG), revealed a significant difference between the K_m values of normal and variant HexA, while no difference was found when the sulfated substrate MUG-6-sulfate (MUGS), which is specific for the α-subunit active site, was used. The V_max values for both substrates were significantly lower in extracts from B1 variant cells than in control extracts, implying a reduced enzyme level in the variant cells. A noncompetitive inhibitor of the reaction with MUGS, N-acetylglucosamine (NAG), induced a significant inhibition (30%) in the mutant cells only. When MUG was used as substrate, variant HexA was found to be more heat stable (T₅₀ = 170 min) than normal HexA (T₅₀ = 65 min). Furthermore, the mutant cell preparation differed from control in the relation between Hex thermosensitivity and protein concentration in the reaction. Two new mutations were identified in exon 5 of the HexA gene: a C⁴⁹⁶ to G transversion, which produced an Arg¹⁶⁶→ Gly alteration and a deletion of C⁴⁹⁸ which generated a shift in the reading frame. The patient was a heterozygote for both mutations even though her parents are first cousins. There is no evidence as yet which of these mutations accounts for the B1 phenotype.