Identification of an Active Acidic Residue in the Catalytic Site of β-Hexosaminidase

Abstract

Human beta-hexosaminidases A and B (EC 3.2.1.52) are dimeric lysosomal glycosidases composed of evolutionarily related alpha and/or beta subunits. Both isozymes hydrolyze terminal beta-linked GalNAc or GlcNAc residues from numerous artificial and natural substrates; however, in vivo GM2 ganglioside is a substrate for only the heterodimeric A isozyme. Thus, mutations in either gene encoding its alpha or beta subunits can result in GM2 ganglioside storage and Tay-Sachs or Sandhoff disease, respectively. All glycosyl hydrolases ae believed to have one or more acidic residues in their catalytic site. We demonstrate that incubation of hexosaminidase with a chemical modifier specific for carboxyl side chains produces a time-dependent loss of activity, and that this effect can be blocked by the inclusion of a strong competitive inhibitor in the reaction mix. We hypothesized that the catalytic acid residue(s) should be located in a region of overall homology and be invariant within the aligned deduced primary sequences of the human alpha and beta subunits, as well as hexosaminidases from other species, including bacteria. Such a region is encoded by exons 5-6 of the HEXA and HEXB genes. This region includes beta Arg211 (invariant in 15 sequences), which we have previously shown to be an active residue. This region also contains two invariant and one conserved acidic residues. A fourth acidic residue, Asp alpha 258, beta 290, in exon 7 was also investigated because of its association with the B1 variant of Tay-Sachs disease. Conservative substitutions were made at each candidate residue by in vitro mutagenesis of a beta cDNA, followed by cellular expression. Of these, only the beta Asp196Asn substitution decreased the kcat (350-910-fold) without any noticeable effect on the K(m). Mutagenesis of either beta Asp240 or beta Asp290 to Asn decreased kcat by 10- or 1.4-fold but also raised the K(m) of the enzyme 11- of 3-fold, respectively. The above results strongly suggest that beta Asp196 is a catalytic acid residue in beta-hexosaminidase.

Aspartate 313 in the Streptomyces plicatusHexosaminidase Plays a Critical Role in Substrate-assisted Catalysis by Orienting the 2-Acetamido Group and Stabilizing the Transition State

“…This rate reduction is considerably less than the 560,000-fold reduction observed for the SpHex D313N variant studied here. These investigators, studying this difficult system, indicate that the enzyme preparation is ϳ99.99% free of wild-type contamination, a marked improvement over earlier studies with the same enzyme (36). However, assuming 0.01% contamination and a completely inactive variant enzyme the greatest possible rate reduction that can be measured is 10,000-fold and is very close to the rate reduction that these workers report.…”

A Pro504 → Ser Substitution in the β-Subunit of β-Hexosaminidase A Inhibits α-Subunit Hydrolysis of GM2Ganglioside, Resulting in Chronic Sandhoff Disease

“…5C) substitutions were examined. The latter substitution results in only monomeric, precursor ␤-chains in transfected cells (23) and thus serves as a control for ER retention (39,44). Quantitative measurements of the green versus yellow (overlapping of the red and green labels in the cytoplasm, i.e.…”

“…The mutation was verified by DNA sequencing. A construct encoding an Asp 208 3 Asn substitution in the ␤-cDNA insert of pEFNEO has previously been reported (23). In permanently transfected CHO cells, this construct produces only soluble, monomeric, precursor ␤-subunits (23).…”

“…This hypothesis has been demonstrated to be correct for substitutions at either residue based on mutational and expression studies of the aligned ␤-residues, i.e. ␤-analogs, ␤-Arg 211 (21,22) and ␤-Asp 290 (23), and on molecular modeling of human Hex using the structure of bacterial chitobiase (24).…”

Molecular cloning and sequence analysis of an ascidian egg β‐N‐acetylhexosaminidase with a potential role in fertilization

“…Alignment of the amino acid sequence of these ␤-hexosaminidases is shown in Figure 2. All of the residues, which are thought to form the active site of the subunits, suggested either from analyses of mutant human proteins (Brown & Mahuran 1991;Liessem et al 1995;Tse et al 1996;Fernandes et al 1997) or from structural analysis of a member of the glycosyl hydrolase family 20 (Serratia marcescens chitobiase; Tews et al 1996), are conserved in P. mammillata. We found five potential N-glycosylation sites in the P. mammillata ␤-hexosaminidase.…”