GM2 gangliosidosis B1 variant: biochemical and molecular characterization of hexosaminidase A.
The biochemical properties of hexosaminidase A (HexA) and the coding sequence of the alpha-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 beta-subunit specific substrate, 4-methylumbelliferyl-beta-D-N-acetylglucosamine (MUG), revealed a significant difference between the Km values. of normal and variant HexA, while no difference was found when the sulfated substrate MUG-6-sulfate (MUGS), which is specific for the alpha-subunit active site, was used. The Vmax 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.
Peleg
L
L
Genetic Institute, Chaim Sheba Medical Center, Tel Hashomer, Israel.
Meltzer
F
F
Karpati
M
M
Goldman
B
B
D001483:Base Sequence; D002478:Cells, Cultured; D005091:Exons; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D054818:Hexosaminidase A; D006801:Humans; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D013661:Tay-Sachs Disease / Q000201:enzymology* / Q000209:etiology / Q000235:genetics; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology*
D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases