Fluorinated carbohydrates as probes of mechanism and specificity in glycosyl transferases

The Compounds 2-deoxy-2-fluoro-β-D-glucosyl fluoride (1), 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2), 2-deoxy-2-fluoro-β-D-galactosyl fluoride (3) and 2-deoxy-2-fluoro-β-D-mannosyl fluoride (4) were all found to be potent covalent inhibitors of β-glucosidase from Alcaligenes faecalis (pABG5 β-glucosidase), which function through the accumulation of a stable glycosyl-enzyme intermediate. The mechanism of action for these inhibitors was investigated and found to be both specific and active site directed, involving a 1:1 stoichiometric formation of an enzyme inhibitor complex. Investigation of the pre-steady state kinetics for the inhibition reaction provided values for the rates of formation (kon) and hydrolysis (koff) of the glycosylated-enzyme intermediate. The values of kon determined in this manner are 5.9, 25, 3.6 and 5.6 min.⁻¹ for compounds 1,2,3 and 4 respectively, while the values of koff were found to be much smaller; 1.5x10⁻⁵, 5.4 x 10⁻³ and 1.0 x 10⁻³ min.⁻¹, respectively for the intermediates formed from compounds 1, 3 and 4. The isolated intermediates were also found to be competent in the catalysis of transglycosylation reactions and evidence for the participation of a specific binding site for the acceptor compound in this process was gained. The results of these kinetic experiments were corroborated by data obtained from ¹⁹F-NMR spectra of the glycosyl-enzyme intermediate and by isolation and subsequent characterization of the transglycosylation products. The stereochemistry of the inhibition reaction was investigated by a simple ¹⁹F-NMR experiment, and was found to be consistent with a double displacement reaction mechanism, as would be expected from the generally accepted reaction mechanism for this type of enzyme. The compound D-glucal was found to be a substrate for pABG5 β-glucosidase (kcat = 2.28 min.⁻¹ Km = 0.85 mM). Hydration of this compound by pABG5 β-glucosidase in deuterated buffers demonstrated that the double bond of D-glucal was deuterated stereo-specifically from below the α-face. The compound 2-fluoro-D-glucal was found to be a weak competitive inhibitor (Ki = 30 mM) of pABG5 β-glucosidase. Glycogen phosphorylase catalyses the reversible phosphorolysis of glycogen. A series of deoxy analogues of the enzymes natural substrate, α-D-glucose 1-phosphate have been prepared and along with a number of deoxyfluoro analogues tested as substrates. All were found to act as substrates but at exceedingly slow rates. The large rate reductions when compared with the normal substrate can be attributed to a deleterious combination of electronic and binding effects in the modified substrates reducing the stability of the enzymic transition states. A linear free energy relationship between kcat and the first order rate constant for the acid catalysed hydrolysis of the same series of deoxy and deoxyfluoro glucopyranosyl phosphates was demonstrated, suggesting similar transition states for the two reactions and implicating an oxocarbonium ion-like transition state in the enzymic reaction. The binding data obtained from the steady state kinetics of these analogues suggests that hydrogen bonding interactions are qualitatively conserved in the glucopyranose binding site during the T- to R-state conformational transition of the enzyme and that interactions between the enzyme and the hydroxyl groups at the 3- and 6-positions of the glucopyranose ring of the substrate are potentially important for stabilization of the enzymic transition state. The specificity of the substrate phosphate binding site has been probed using the compounds 2-deoxy-2-fluoro-α-D-glucopyranosyl phosphate (5), (1-deoxy-α-D-glucopyranosyl) methylphosphonate (6) and 2-deoxy-2-fluoro-α-D-glucopyranosyl phosphofluoridate (7). The results suggested that phosphorylase b can bind both the monanionic and dianionic forms of its substrate with approximately equal affinity. NMR studies of the ternary enzyme-ligand complexes formed with glycogen phosphorylase b and 5 or 6 indicated that no proton donation occurred in the ground-state active site complex. A preliminary investigation into the ability of the cellulase complex from a number of different wood-degrading fungi to hydrolyse p-nitrophenyl β-glucoside has been carried out. This work is aimed at producing environmentally safe fungicides, which are activated by the β-glucosidase component of the cellulase complex in these organisms and this study was carried out in conjunction with Forintek Canada.Science, Faculty ofChemistry, Department ofGraduat