Substituted aryl glycosides as probes of the mechanism of spontaneous ad enzyme-catalyzed glycoside hydrolysis

• Main Author: Namchuck, Mark N.
• Language: English
• Published: 2008
• Online Access: http://hdl.handle.net/2429/1910

A series of mono-substituted deoxy and deoxyfluoro 2',4'-dinitrophenyl P-D-glycopyranosides (DNP glycosides) was synthesized and used to probe the mechanisms of spontaneous and enzyme-catalyzed hydrolysis of 0-glycosides. The relative rates of spontaneous hydrolysis of the DNP glucosides followed the trend 4-deoxy > 3-deoxy - 6-deoxy > parent > 6-deoxy-6-fluoro > 3-deoxy-3-fluoro > 4-deoxy-4-fluoro > 2-deoxy-2-fluoro. These relative rates can be rationalized on the basis of the stabilities of the oxocarbonium ions generated at the transition state, which appear to be principally a function of field effects (as predicted by the Kirkwood-Westheimer hypothesis) exerted by the ring substituents at 05 and Cl. Differences in the rates of hydrolysis between different series of hexopyranosides could not be explained simply on the basis of field effects and likely arise from several factors. Based on the large differences in the values of the activation entropy and enthalpy for hydrolysis of the analogously substituted galactosides and glucosides, it is suggested that differences in the hydration structure of these glycosides at the transition state could be contributing to the observed rate differences. The role of non-covalent interactions between the enzyme active site and the hydroxyl groups of the substrate glycone in the mechanism of Agrobacterium faecalis r3-glucosidase, as well as the amount of charge generated at the transition states for the enzymic reaction, were probed using the same DNPglycosides. Both pre-steady state and steady state kinetic analysis of the hydrolysis of the DNPglycosides by Agrobacterium 0-glucosidase was performed to investigate the effect of the ring substitutions on each of the steps in the enzyme mechanism. Non-covalent enzyme/substrate interactions at the 2, 3 and 6 positions contributed respective binding energies of at least 18, 7 and 3 k.1/mol , to stabilization of the transition state for glycosylation of the enzyme. Binding effects at the transition state for deglycosylation were similar. The interaction at the 4 position is unique in that it appears to stabilize the transition state for glycosylation to a greater extent than the transition state for glycosylation. Evidence is also presented to support an earlier proposal (Kempton and Withers (1992), Biochemistry 31, 9961) that the transition states for glycosylation and deglycosylation of the enzyme may differ in the amount of positive charge generated, with deglycosylation being significantly more oxocarboniumion like. The rates of enzymic hydrolysis obtained in this study were then used to investigate the mode of action of castanospermine and 1-deoxynojirimycin as non-covalent inhibitors of Agrobacterium 5-glucosidase. Dissociation constants (KO were determined for a series of mono-substituted castanospermines and 1-deoxynojirimycins,and the logarithm of those dissociation constants correlated with the logarithm of the rate constants for the Agrobacterium P-glucosidase-catalyzed hydrolysis of the analogously substituted DNPglycosides. In all cases, these parameters correlated poorly, regardless of whether Ki for the inhibitor was compared with Kd, kcat or kcat/Km for the analogoussubstrates. Based on these data, it is unlikely that the tight binding of castanospermine and 1-deoxynojirimycin to this enzyme is due to their being transition state analogues. Binding of these inhibitors is likely better described as 'fortuitous' wherein the positively charged amines are binding to the anionic active site of an enzyme which has evolved to bind a polyhydroxylated, positively charged transition state.