Direct Determination of the Substrate Specificity of the α-Active Site in Heterodimeric β-Hexosaminidase A

The β-hexosaminidase isozymes are produced through the combination of α and β subunits to form any one of three active dimers (monomeric subunits are not functional). Heterodimeric hexosaminidase A (αβ) is the only isozyme that can hydrolyze GM2 ganglioside in vivo, requiring the presence of the GM2 activator protein. Hexosaminidase S (αα) exists but is not considered a physiological isozyme. Although hexosaminidase B (ββ) is present in normal human tissues, it has no known unique function in vivo. However, a unique function for the β-active site present in both hexosaminidase A and B has been indicated in a previous study of the various substrate specificities of the homodimeric forms of hexosaminidase (S and B). It was concluded that the α-active site is only able to efficiently hydrolyze negatively charged substrates, and the β-active site is only able to hydrolyze neutral substrates. When this model of nonoverlapping α- and β-substrates is extrapolated to heterodimeric hexosaminidase A, it has a major effect on the interpretation of recent results relating to the mode of action of the GM2 activator protein. In this report, we directly examine these substrate specificities using a novel form of hexosaminidase A containing an inactive β subunit, produced in permanently transfected CHO cells. We demonstrate that, whereas the β-active site has the same substrate specificities in either its A-heterodimeric or B-homodimeric forms, the α-active site in the A-heterodimer has different kinetic parameters than the α-active site in the S-homodimer. We conclude that the α and β subunits in hexosaminidase A participate equally in the hydrolysis of neutral substrates.