Identification of the 6-sulfate binding site unique to alpha-subunit-containing isozymes of human beta-hexosaminidase

In humans, beta -hexosaminidase A (alpha beta) is required to hydrolyze GM2 ganglioside. A deficiency of either the alpha- or beta -subunit leads to a severe neurological disease, Tay-Sachs or Sandhoff disease, respectively. In mammals beta -hexosaminidase B (beta beta) and S (alpha alpha) are other major and minor isozymes. The primary structures of the alpha- and beta -s ubunits are 60% identical, but only the alpha -containing isozymes can effi ciently hydrolyze beta -linked GlcNAc-6-SO4 from natural or artificial subs trates. Hexosaminidase has been grouped with glycosidases in family 20. A m olecular model of the active site of the human hexosaminidase has been gene rated from the crystal structure of a family 20 bacterial chitobiase. We no w use the chitobiase structure to identify residues close to the carbon-6 o xygen of NAG-A, the nonreducing beta -GlcNAc residue of its bound substrate . The chitobiase side chains in the best interactive positions align with a lpha -Asn(423)Arg(424) and beta -Asp(453)Leu(454). The change in charge fro m positive in alpha to negative in beta is consistent with the lower K-m of hexosaminidase S, and the much higher K-m and lower pH optimum of hexosami nidase B, toward sulfated versus unsulfated substrates. In vitro mutagenesi s, CHO cell expression, and kinetic analyses of an alpha Arg(424)Lys hexosa minidase 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 unaffect ed. When alpha Asn(423) was converted to Asp, again only the K-m for the su lfated substrate was changed, increasing by 6-fold. Neutralization of the c harge on alpha Arg(424) by substituting Gin produced a hexosaminidase S wit h a K-m decrease of 3-fold and a V-max increased by 6-fold for the unsulfat ed substrate, parameters nearly identical to those of hexosaminidase B at p H 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 he xosaminidase B obtained at pH 3.0, where its beta Asp(453) becomes protonat ed.