Characterization of the active site of [beta]-hexosaminidase

Human hexosaminidases (EC 3.2.1.52) are lysosomal hydrolases that cleave terminal non-reducing β-1,4-linked N-acetylhexosamines from natural substrates, e.g., glycolipids, glycoproteins, gangliosides and glycosaminoglycans and artificial substrates, e.g., methylumbelliferyl-β-1,4-GlcNAc (MUG) and methylumbelliferyl-β-1,4-GlcNAc-6-sulfate (MUGS). There are three human β-hexosaminidase isozymes which are composed of all possible dimeric combinations of α- and/or β-subunits, i.e., A (αβ), B (ββ), and S (αα). The amino acid sequences of the two subunits are 60% identical. Although dimerization is required for activity, kinetic studies have shown that the α- and β-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 β-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 α-subunit to bind negatively charged substrates is located within residues α132–283. In addition, the carboxy-terminal section from the β-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 α and β 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 βArg 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., βAsp196, βAsp 208, βAsp240 and βGlu253. Residue βAsp290, located in exon 7, was also investigated in this study because its aligned residue in the α-subunit, αAsp 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 β-cDNA, followed by cellular expression. Of these, only the βAsp196Asn substitution decreased the kcat (350–910-fold) without any noticeable effect on the Km. Mutagenesis of either βAsp240 or βAsp290, 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, βAsp 240 and βAsp290, were not catalytic residues but are likely involved in substrate binding. The study also demonstrates that βAsp 208 is involved in the initial folding or dimer formation of the β-subunits, These data strongly suggest that the residue, βAsp196, is a catalytic acid residue in β-hexosaminidase.