Identification of the 6-Sulfate Binding Site Unique to α-Subunit-Containing Isozymes of Human β-Hexosaminidase^†

In humans, β-hexosaminidase A (αβ) is required to hydrolyze GM2 ganglioside. A deficiency of either the α- or β-subunit leads to a severe neurological disease, Tay-Sachs or Sandhoff disease, respectively. In mammals β-hexosaminidase B (ββ) and S (αα) are other major and minor isozymes. The primary structures of the α- and β-subunits are 60% identical, but only the α-containing isozymes can efficiently hydrolyze β-linked GlcNAc-6-SO₄ from natural or artificial substrates. Hexosaminidase has been grouped with glycosidases in family 20. A molecular model of the active site of the human hexosaminidase has been generated from the crystal structure of a family 20 bacterial chitobiase. We now use the chitobiase structure to identify residues close to the carbon-6 oxygen of NAG-A, the nonreducing β-GlcNAc residue of its bound substrate. The chitobiase side chains in the best interactive positions align with α-Asn⁴²³Arg⁴²⁴ and β-Asp⁴⁵³Leu⁴⁵⁴. The change in charge from positive in α to negative in β is consistent with the lower K_m of hexosaminidase S, and the much higher K_m and lower pH optimum of hexosaminidase B, toward sulfated versus unsulfated substrates. In vitro mutagenesis, CHO cell expression, and kinetic analyses of an αArg⁴²⁴Lys hexosaminidase S detected little change in V_max but a 2-fold increase in K_m for the sulfated substrate. Its K_m for the nonsulfated substrate was unaffected. When αAsn⁴²³ was converted to Asp, again only the K_m for the sulfated substrate was changed, increasing by 6-fold. Neutralization of the charge on αArg⁴²⁴ by substituting Gln produced a hexosaminidase S with a K_m decrease of 3-fold and a V_max increased by 6-fold for the unsulfated substrate, parameters nearly identical to those of hexosaminidase B at pH 4.2. As well, for the sulfated substrate at pH 4.2 its K_m was increased 9-fold and its V_max decreased 1.5-fold, values very similar to those of hexosaminidase B obtained at pH 3.0, where its βAsp⁴⁵³ becomes protonated.