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

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Abstract
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 Km of hexosaminidase S, and the much higher Km 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 Vmax but a 2-fold increase in Km for the sulfated substrate. Its Km for the nonsulfated substrate was unaffected. When αAsn423 was converted to Asp, again only the Km 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 Km decrease of 3-fold and a Vmax 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 Km was increased 9-fold and its Vmax decreased 1.5-fold, values very similar to those of hexosaminidase B obtained at pH 3.0, where its βAsp453 becomes protonated.