Human β-hexosaminidase A (Hex A) (αβ) is composed of two subunits whose primary structures are ~60% identical. Deficiency of either subunit results in severe neurological disease due to the storage of GM2 ganglioside; Tay-Sachs disease, α deficiency, and Sandhoff disease, β deficiency. Whereas both subunits contain active sites only the α-site can efficiently bind negatively charged 6-sulfated hexosamine substrates and GM2 ganglioside. We have recently identified the αArg 4 2 4 as playing a critical role in the binding of 6-sulfate-containing substrates, and βAsp 4 5 2 as actively inhibiting their binding. To determine if these same residues affect the binding of the sialic acid moiety of GM2 ganglioside, an αArg 4 2 4 Gln form of Hex A was expressed and its kinetics analyzed using the GM2 activator protein:[ 3 H]-GM2 ganglioside complex as a substrate. The mutant showed a ~3-fold increase in its K m for the complex. Next a form of Hex B (ββ) containing a double mutation, βAspLeu 4 5 3 AsnArg (duplicating the α-aligning sequences), was expressed. As compared to the wild type (WT), the mutant exhibited a >30-fold increase in its ability to hydrolyze a 6-sulfated substrate and was now able to hydrolyze GM2 ganglioside when the GM2 activator protein was replaced by sodium taurocholate. Thus, this α-site is critical for binding both types of negatively charge substrates.