Identification of two amino acid residues which determine the substrate specificity of human β-D-N-acetylhexosaminidase A

Abstract

β-D-N-acetylhexosaminidases A (Hex A, αβ) and B (Hex B, ββ) cleave N-acetylglucosamine and N-acetylgalactosamine termini of glycoconjugates. Hex B hydrolyzes neutral substrates whereas Hex A also hydrolyzes electronegative substrates, including GM₂ ganglioside, which accumulates in the neurons of patients with Tay-Sachs disease (TSD). We hypothesized that enzyme-substrate electrostatic interactions influence substrate specificities of the two isozymes. Among seven positively charged candidate residues in Hex A, at which substitution for the homolgous β residue was performed, only the α424R→L mutation resulted in loss of activity toward the electronegative substrate 4-methylumbellifery-N-acetylglucosamine-6-sulfate (4MUGS). The substitution L→R at the homolgous β position 453 increased Hex B activity to 4MUGS 5-fold. αR453 projects into the α-subunit substrate cavity opposite three active site amino acids. The adjacent residue, βD452, may repel negatively charged substrates. Double substitution, βL453R and βD452→N (the α-subunit homologue), increases 4MUGS hydrolysis by 22-fold relative to wild type Hex B. These results indicate that the homology model for hexosaminidase gives an accurate picture of the active site region and may furnish other candidate residues to test as determinants of the unique substrate specificity of Hex A.