The β-hexosaminidases (Hex) catalyze the cleavage of terminal amino sugars on a broad spectrum of glycoconjugates. The major Hex isozymes in humans, Hex A, a heterodimer of α and β subunits (αβ), and Hex B, a homodimer of β subunits (ββ), have different substrate specificities. The β subunit (HEXB gene product), hydrolyzes neutral substrates. The α subunit (HEXA gene product), hydrolyzes both neutral and charged substrates. Only Hex A is able to hydrolyze the most important natural substrate, the acidic glycolipid GM2 ganglioside. Mutations in the HEXA gene cause Tay-Sachs disease (TSD), a GM2 ganglioside storage disorder. We investigated the role of putative active site residues Asp-α258, Glu-α307, Glu-α323, and Glu- α462 in the α subunit of Hex A. A mutation at codon 258 which we described was associated with the TSD B1 phenotype, characterized by the presence of normal amounts of mature but catalytically inactive enzyme. TSD-B1 mutations are believed to involve substitutions of residues at the enzyme active site. Glu-α307, Glu-α323, and Glu-α462 were predicted to be active site residues by homology studies and hydrophobic cluster analysis. We used site-directed mutagenesis and expression in a novel transformed human fetal TSD neuroglial (TSD-NG) cell line (with very low levels of endogenous Hex A activity), to study the effects of mutation at candidate active site residues. Mutant HEXA cDNAs carrying conservative or isofunctional substitutions at these positions were expressed in TSD-NG cells. αE323D, αE462D, and αD258N cDNAs produced normally processed peptide chains with drastically reduced activity toward the α subunit-specific substrate 4MUGS. The αE307D cDNA produced a precursor peptide with significant catalytic activity. Kinetic analysis of enzymes carrying mutations at Glu-α323 and Asp-α258 (reported earlier by Bayleran, J., Hechtman, P., Kolodny, E., and Kaback, M. (1987) Am. J. Hum. Genet. 41,532-548) indicated no significant change in substrate binding properties. Our data, viewed in the context of homology studies and modeling, and studies with suicide substrates, suggest that Glu-α323 and Asp. α258 are active site residues and that Glu-α323 is involved in catalysis
