UBC Theses and Dissertations
The use of inhibitors to stabilize folded conformations of enzymes Reid, Stephen P.
Tay Sachs and Gaucher diseases are lysosomal storage disorders that are caused by deficiencies in the lysosomal enzymes β-hexosaminidase A and glucocerebrosidase, respectively. The glycolipid substrates of deficient β-hexosaminidase A and glucocerebrosidase accumulate in lysosomal neurons, resulting in neurodegenerative disorders. The enzyme deficiencies are caused by heritable amino acid point mutations that result in unstable and potentially misfolded enzymes. The mutant enzymes, although catalytically active in many cases, are destroyed by cellular quality control mechanisms in the endoplasmic reticulum (ER). A potential therapy for adult chronic Tay Sachs disease and Gaucher disease is the use of enzyme inhibitors as chemical chaperones. To act as a chemical chaperone the enzyme-specific inhibitor must stabilize the folded conformation of the enzyme and thus prevent enzyme destruction by cellular quality control mechanisms. The enzyme/inhibitor complex can then be transferred from the E R to the lysosome where the substrate concentration is significantly high that some enzyme activity is restored. In the chronic adult disease forms, only 10 % of normal enzyme activity is required for a patient to become asymptomatic. A competitive inhibitor for β-hexosaminidase A, NAG-thiazoline, was synthesized and shown to be a potent inhibitor o f β-hexosaminidase A and the related enzyme β-hexosaminidase B, with Ki values of 270 nM and 190 nM , respectively. Chemical denaturation studies showed that NAG-thiazoline stabilizes a bacterial hexosaminidase from Streptomyces plicatus (Sp. Hex.) and human P-hexosaminidase B against guanidine hydrochloride denaturation. Thermal denaturation studies showed that NAG-thiazoline stabilizes Sp. Hex., β-hexosaminidase B, and β- hexosaminidase A against thermal denaturation. These results provide a chemical explanation for recent results from collaborator Dr. Mike Tropak (The Hospital for Sick Children, Toronto), which showed that NAG-thiazoline increases Phexosaminidase A activity in Tay Sachs fibroblasts. A mechanism based inactivator for glucocerebrosidase, 2-deoxy-2-fluoro- β-Dglucopyranosyl fluoride, was synthesized and shown to stabilize glucocerebrosidase against guanidine hydrochloride denaturation. N-Octyl-l-epivalienamine was synthesized and shown to be a potent competitive inhibitor o f glucocerebrosidase with a Ki value of 75 nM and to stabilize glucocerebrosidase against thermal denaturation. These results show that 2-deoxy-2-fluoro- β-D-glucopyranosyl fluoride and N-octyl-l-epivalienamine stabilize the folded conformation of glucocerebrosidase and may act as chemical chaperones in therapies for Gaucher disease. An additional study was performed in which the synthetic capacity of a glycosynthase developed from a β-glucuronidase from Thermotoga maritima was explored. Using α-D-glucopyranuronosyl fluoride or α-D-galactopyranuronosyl fluoride as donors, and pNP-glucoside, pNP-xyloside, and pNP-cellobioside as acceptors, the E476A β-glucuronidase mutant catalyzed the formation of six novel oligosaccharides.
Item Citations and Data