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Glycals as probes for mechanistic studies of glycosylases

University of British Columbia

Several derivatives of D-gluco-heptenitol and D-glucal were synthesized and used to study the reaction mechanisms of three glycosylases (glycosyl mobilizing enzymes): glycogen phosphorylase, J3-glucosidase, and β-N-acetythexosaminidase. l-Fluoro-D-gluco-heptenitol (F₁hept) and 1, 1-difluoro-D-gluco-heptenitol ( F₂hept) acted as competitive inhibitors of glycogen phosphorylase b, and collaborative X-ray crystallographic studies revealed that both F₁hept and F₂hept bind to the active site. Furthermore, F₁hept and phosphate bind simultaneously, allowing crystallographic investigation of a stable ternary complex. Various glycals were examined as potential substrates (catalytically hydrated by the enzyme), or as potential inhibitors or inactivators (of the hydrolysis of a glucoside substrate), of Agrobacterium β -glucosidase. Both heptenitol and methylglucal acted as substrates of this enzyme. Enzymatic protonation of the double bond of methylglucal occurred from below the ring. F₁hept and Fhept acted as noncompetitive (or uncompetitive) inhibitors, as did heptenitol (at concentrations < 8 mM). α, β -Unsaturated glucals, which might act as Michael acceptors for a nucleophilic residue in the active site of a glycosylase, were investigated as a new class of potential inactivators of Agrobacterium β -glucosidase. 1-Nitroglucal functioned as a time-dependent inactivator, probably as a result of a Michael addition reaction between 1-nitroglucal and a nucleophilic residue in the active site. However, protein mass spectrometry revealed that in most cases more than one equivalent of l-nitroglucal bound to the enzyme. Unfortunately, other α, β -unsaturated glucals that were examined [1-cyano-, 1-(methyl carboxylate)-, sodium 1-(carboxylate)-, and 2-cyano- derivatives of glucal] only acted as reversible inhibitors. Three β N-acetythexosaminidases (human placenta, jack bean, and bovine kidney) were also studied, and shown to hydrolyze an N-acetylglucosaminide substrate with net retention of anomeric configuration. All three enzymes hydrated 2-acetamido-D-glucal, yielding N-acetyl-D-glucosamine as the product in each case. This is the first time that proton transfer has been shown to occur from the top face during the hydration of a glycal by a ‘retaining’ β -glycosidase. 2-Acetamido-D-glucal bound tightly to the human, bovine, and jack bean enzymes, with K₁ values of 8.5, 25, and 29 M, respectively.

Thesis/Dissertation

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2009-06-04

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http://hdl.handle.net/2429/8803

Chemistry

University of British Columbia

UBCV

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