GM2 GANGLIOSIDOSIS B1 VARIANT - BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF HEXOSAMINIDASE-A

The biochemical properties of hexosaminidase A (HexA) and the coding s equence of the alpha-subunit were examined in a patient of Syrian ance stry with the B1 form of Tay-Sachs disease (TSD). The bio chemical cha racteristics of the variant HexA suggest that both active sites are af fected by the mutation(s). Kinetic studies with the beta-subunit speci fic substrate, 4 methylumbelliferyl-beta-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 substra te MUG-g-sulfate (MUGS), which is specific for the alpha-subunit activ e site, was used. The V-max values for both substrates were significan tly lower in extracts from BI 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), induc ed a significant inhibition (30%) in the mutant cells only. When MUG w as used as substrate, variant HexA was found to be more heat stable (T -50 = 170 min) than normal HexA (T-50 = 65 min). Furthermore, the muta nt cell preparation differed from control in the relation between Hex thermosensitivity and protein concentration in the reaction. Two new m utations were identified in exon 5 of the HexA gene: a C-496 to G tran sversion, which produced an Arg(166)--> Gly alteration and a deletion of C-498 which generated a shift in the reading frame. The patient was a heterozygote for both mutations even though her parents are first c ousins. There is no evidence as yet which of these mutations accounts for the B1 phenotype. (C) 1995 Academic Press, Inc.