The alpha- and/or beta-subunits of human beta-hexosaminidase A (alphabeta) and B (betabeta) are similar to60% identical. In vivo only beta-hexosaminidase A can utilize GM2 ganglioside as a substrate, but requires the GM2 activator protein to bind GM2 ganglioside and then interact with the enzyme, placing the terminal GalNAc residue in the active site of the a-subunit. A model for this interaction suggests that two loop structures, present only in the alpha-subunit, may be critical to this binding. Three amino acids in one of these loops are not encoded in the HEXB gene, while four from the other are removed posttranslationally from the pro-beta-subunit. Natural substrate assays with forms of hexosaminidase A containing mutant a-subunits demonstrate that only the site that is removed from the beta-subunit during its maturation is critical for the interaction. Our data suggest an unexpected biological role for such proteolytic processing events. (C) 2004 Elsevier Inc. All rights reserved.