Mechanistic studies of retaining α-glycosidases

by Ran Zhang

Institution: University of British Columbia
Department: Chemistry
Degree: PhD
Year: 2010
Record ID: 1885565
Full text PDF: http://hdl.handle.net/2429/27445


The majority of retaining α-glycosidases are believed to adopt the classical double displacement mechanism to catalyze their reactions, which features a catalytic nucleophilic residue, a general acid/base residue, two oxocarbenium-ion like transition states and one covalent glycosyl-enzyme intermediate. In my thesis, the catalytic mechanisms of three retaining alpha-glycosidases were investigated in detail as follows. HPA is an enzyme which is responsible for hydrolyzing starch into shorter oligosaccharides. Several 2-deoxy-2,2-dihalo maltosyl chlorides were synthesized and tested as potential mechanism-based inhibitors of HPA, in the hope of trapping its covalent glycosyl-enzyme intermediate for crystallographic studies. Unfortunately, none of newly-synthesized compounds could cause time-dependent inactivation of HPA. By employing our newly developed in situ elongation strategy, 5-fluoro-α-D-glucopyranosyl fluoride and 5-fluoro-β-L-idopyranosyl fluoride showed kinetic behavior consistent with the proposed in situ elongation-inactivation process, allowing the trapping and further kinetic and structural analysis of the covalent intermediate of HPA. These structures provide interesting mechanistic insights into the catalytic mechanism of HPA. TreS is an enzyme which catalyzes the reversible interconversion of maltose and trehalose. 5-Fluoro glycosyl fluorides were shown to be mechanism-based inhibitors of this enzyme by accumulating the covalent glycosyl-enzyme intermediate. The trapped intermediate was subjected to protease digestion followed by MS analysis of the resultant peptides to identify the catalytic nucleophile residue as D230. The inability of TreS to carry out transglycosylation reactions onto exogenously added acceptors establishes the intramolecular nature of the rearrangement reaction, consistent with previous studies on other TreS enzymes. All studies support a double displacement mechanism involving an intramolecular “glucose flipping” step as the catalytic mechanism of this enzyme. SpGH101 is an enzyme which specifically removes an O-linked disaccharide Gal-β-1,3-GalNAc-α from glycoproteins. Using the recently solved 3-dimensional structure of this protein as a guide, we carried out a detailed mechanistic investigation of this retaining α-glycosidase using a combination of synthetic and natural substrates. Based on a model of the substrate complex of SpGH101, we proposed D764 and E796 as the nucleophile and general acid/base residues, respectively. These roles were confirmed by kinetic and mechanistic analysis of mutants at those positions using synthetic substrates and anion rescue experiments.