Identification of candidate active site residues in lysosomal beta-hexosaminidase A

The beta-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 alpha and beta subunits (alpha beta), and Hex B, a homodimer of beta subunits (beta beta), have different substrate specificities. The beta subunit (HEXB gene product), hydrolyzes neutral substrates. The alpha 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 GM(2) ganglioside. Mutations in the HEXA gene cause Tay-Sachs disease (TSD), a GM(2) ganglioside storage disorder. We investigated the role of putative active site residues Asp-alpha 258, Glu-alpha 307, Glu-alpha 323, and Glu-alpha 462 in the alpha 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-alpha 307, Glu-alpha 323, and Glu-alpha 462 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. alpha E323D, alpha E462D, and alpha D258N cDNAs produced normally processed peptide chains with drastically reduced activity toward the alpha subunit-specific substrate 4MUGS. The alpha E307D cDNA produced a precursor peptide with significant catalytic activity. Kinetic analysis of enzymes carrying mutations at Glu-alpha 323 and Asp-alpha 258 (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 substrate, suggest that Glu-alpha 323 and Asp-alpha 258 are active site residues and that Glu-alpha 323 is involved in catalysis.