A single site in human β-hexosaminidase A binds both 6-sulfate-groups on hexosamines and the sialic acid moiety of GM2 ganglioside

Abstract

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 424 as playing a critical role in the binding of 6-sulfate-containing substrates, and βAsp 452 as actively inhibiting their binding. To determine if these same residues affect the binding of the sialic acid moiety of GM2 ganglioside, an αArg 424Gln form of Hex A was expressed and its kinetics analyzed using the GM2 activator protein:[ 3H]-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 453AsnArg (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.