Structure-function relationships in human ß-hexosaminidase

grantor: University of TorontoHuman ß-hexosaminidase (Hex) isozymes are dimeric enzymes composed of à and/or ß subunits, i.e. Hex A (Ãß), Hex B (ßß) and Hex S (ÃÃ). Only Hex A has a unique in vivo function, 'i.e'., hydrolysis of ganglioside GM2. Thus, mutations in the either à or ß subunit of Hex A can result in ganglioside storage and two major forms of GM2 gangliosidosis, ' i.e'. Tay-Sachs or Sandhoff diseases, respectively. The goal of this thesis is to study structure-function relationship in human Hex. The first project was to characterize a novel missense mutation (ßPro504Ser) associated with the chronic form of Sandhoff disease using both the patients' and transfected cells. Biochemical studies of mutant Hex A demonstrated that this substitution directly affects the ability of Hex A to hydrolyze its natural substrate and also intracellular transport, producing chronic Sandhoff disease. The second project was to examine the validity of the molecular modeling of human Hex based on the X-ray structure of bacterial chitobiase. To achieve this, I first developed a new method for the generation of a C-terminal His6-tagged form of pro-Hex B in transfected cells. Re-examination of ßArg21 I Lys using this highly purified form of mutant Hex B indicated that the substitution resulted in an enzyme with a more than 10-fold increase in its apparent Km and over a 1000-fold decrease in its Vmax. Thus ßArg211 plays a critical role in substrate binding and in orientating the substrate in the active site. Next I used the same approach to analyze the candidate conserved active acidic residues in human Hex B. The ßGlu355GIn substitution reduced Vmax >8000-fold with a slight lowering of the Km, consistent with Glu355 being the catalytic acid group in human Hex. The ßAsp354Asn mutant also had an almost normal 0Km but a 6000-fold Vmax decrease, also conforming with Asp354 functioning as a "base" to aid in the proposed substrate-assisted catalysis. ßAsp241, along with ßAsp240 and ßAsp290, is involved in substrate binding; whereas ßAspl96 is important in the initial folding or dimer formation of the ß-subunits rather than any involvement in its active site. Taken together, I concluded that the chitobiase model is correct in light of the active site residues in human Hex characterized thus far. The studies of these active site residues suggest that human Hex and probably all the Family 20 enzymes have similar structures and a conserved substrate-assisted catalysis mechanism.Ph.D