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

Abstract

In humans, beta-hexosaminidase A (alphabeta) 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 (betabeta) and S (alphaalpha) are other major and minor isozymes. The primary structures of the alpha- and beta-subunits are 60% identical, but only the alpha-containing isozymes can efficiently hydrolyze beta-linked GlcNAc-6-SO(4) 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 beta-GlcNAc residue of its bound substrate. The chitobiase side chains in the best interactive positions align with alpha-Asn(423)Arg(424) and beta-Asp(453)Leu(454). The change in charge from 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 hexosaminidase B, toward sulfated versus unsulfated substrates. In vitro mutagenesis, CHO cell expression, and kinetic analyses of an alphaArg(424)Lys 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 alphaAsn(423) was converted to Asp, again only the K(m) for the sulfated substrate was changed, increasing by 6-fold. Neutralization of the charge on alphaArg(424) 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 betaAsp(453) becomes protonated.

Figure 1

(A) Output of the program RasMol v2.6.1 and the file “1QBB-chito+dinag.pdb” from the Protein Data Base web site. The file contains the coordinates of the crystal structure of bacterial chitobiase with a bound substrate molecule chitobiose [NAG(A)-NAG(B)]. The program allows one to identify residues within a set distance from another moiety. In the picture four residues, shown in “ball-and-stick” format, were identified as having atoms within 3.5 Å of the oxygen atom on carbon-6 of NAG-A, shown in the “space-filling” format (other surrounding residues are shown in the “wire” format). Of these four residues it is apparent that Asp⁶⁷¹ and Phe⁶⁷³ have side chains in the best positions to interact with NAG-A’s O6 atom. (B) Alignment of the area identified in “A” on the basis of the molecular modeling of human Hex from chitobiase (13) with residues in human and mouse α- and β-subunits.

Figure 2

pH optima profiles of purified placental Hex B using MUG or MUGS as a substrate. Florescent units were plotted versus pH.

Figure 3

Kinetic examination of purified placental Hex B using MUGS as a substrate at pH = 3.0 or 4.2. At pH = 3.0, substrate inhibition was noted with MUGS concentrations >3.5 mM (data not shown); thus assays were restricted to less than optimal substrate concentrations. The absolute specific activities (nanomoles of MU per hour per nanogram of Hex B) were plotted versus substrate concentration.

Figure 4

Western blot of transfected CHO cells containing 50 units of MUG activity each: WT, wild-type Hex S; NK, Hex S containing a Arg⁴²⁴ → Lys substitution; DR, Hex S containing a Asn⁴²³ → Asp substitution; NQ, Hex S containing a Arg⁴²⁴ → Gln substitution. Sandhoff is a positive control lane containing lysate from a Sandhoff patient’s fibroblasts, and Neg is the negative control lane containing untransfected CHO cell lysate. The output from Gel Plot Macro of NIH Image 1.60 software is presented as an insert near the bottom of the membrane, and the calculated Areas are reproduced in a larger font size below the scans of each mature αCRM band. Note that the Image program divided the smallest NQ band into two sections. Only the sum of these sections (480) is shown in larger size font below.

Figure 5

Kinetic examinations of the mutant, i.e., WT α-Asn⁴²³-Arg⁴²⁴ → (a) Asn-Lys (NK), (b) Asp-Arg (DR), or (c) Asn-Gln-(NQ), and wild-type (WT) forms of Hex S using (A) MUGS or (B) MUG as a substrate. In these cases Hex S was enriched by ion-exchange chromatography but not purified. Velocity values are given in nanomoles of MU per hour per microgram of total protein.