Characterization of the active site of ß-hexosaminidase

Human hexosaminidases (EC 3.2.1.52) are lysosomal hydrolases that cleave terminal non-reducing ß-1,4-linked N-acetylhexosamines from natural substrates, ' e.g.', glycolipids, glycoproteins, gangliosides and glycosaminoglycans and artificial substrates, 'e.g.', methylumbelliferyl-ß-1,4-GlcNAc (MUG) and methylumbelliferyl-ß-1,4-GlcNAc-6-sulfate (MUGS). There are three human ß-hexosaminidase isozymes which are composed of all possible dimeric combinations of Ã- and/or ß-subunits, 'i.e.', A (Ãß), B (ßß), and S (ÃÃ). The amino acid sequences of the two subunits are 60% identical. Although dimerization is required for activity, kinetic studies have shown that the Ã- and ß-subunits each contain a potential active site which differ slightly in their substrate specificity. 'In vivo', only the heterodimeric Hex A can hydrolyze GM2 ganglioside but to do this it requires a small substrate specific cofactor, the GM2 activator. This thesis deals primarily with the elucidation of active site residue(s) and/or domains which are responsible for the substrate specificities found in different isozymes and the overall activity of human ß-hexosaminidase. To achieve these goals, techniques such as site-directed mutagenesis and the construction of chimeras were employed. My results indicate that the region responsible for the ability of the Ã-subunit to bind negatively charged substrates is located within residues Ã132-283. In addition, the carboxy-terminal section from the ß-chain, residues 316-556, is likely involved in the binding of the GM2-ganglioside/GM2 -activator complex. My hypothesis was that the catalytic residue(s) should be located in the region which has an overall homology but was invariant within the aligned deduced primary sequences of the human Ã and ß subunits, as well as hexosaminidases from other species, including bacteria. Such a region was identified and found to be encoded by exons 5-6 of the 'HEXA ' and 'HEXB' genes. This region also contains ßArg 211 (invariant in all 15 sequences), which was previously shown to be an active residue. Since active acidic residues are present in many glycosidases, I examine candidate acidic active site residues that were located in this region, 'i.e.', ßAsp196, ßAsp 208, ßAsp240 and ßGlu253. Residue ßAsp290, located in exon 7, was also investigated in this study because its aligned residue in the Ã-subunit, ÃAsp 258, has been associated with the B1-variant of Tay-Sachs disease. Conservative substitutions were made at each candidate residue by ' in vitro' mutagenesis of a ß-cDNA, followed by cellular expression. Of these, only the ßAsp196Asn substitution decreased the kcat (350-910-fold) without any noticeable effect on the Km. Mutagenesis of either ßAsp240 or ßAsp290, to Asn decreased kcat by 10- or 1.4-fold, but also raised the K m of the enzyme 11- or 3-fold, respectively. Thus, ßAsp 240 and ßAsp290, were not catalytic residues but are likely involved in substrate binding. The study also demonstrates that ßAsp 208 is involved in the initial folding or dimer formation of the ß-subunits, These data strongly suggest that the residue, ßAsp196, is a catalytic acid residue in ß-hexosaminidase.