R. Sharma et al., Identification of the 6-sulfate binding site unique to alpha-subunit-containing isozymes of human beta-hexosaminidase, BIOCHEM, 40(18), 2001, pp. 5440-5446
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.