Structural, Kinetic, and Thermodynamic Analysis of Glucoimidazole-Derived Glycosidase Inhibitors^,

Abstract

Inhibition of glycosidases has great potential in the quest for highly potent and specific drugs to treat diseases such as diabetes, cancer, and viral infections. One of the most effective ways of designing such compounds is by mimicking the transition state. Here we describe the structural, kinetic, and thermodynamic dissection of binding of two glucoimidazole-derived compounds, which are among the most potent glycosidase inhibitors reported to date, with two family 1 beta-glycosidases. Provocatively, while inclusion of the phenethyl moiety improves binding by a factor of 20-80-fold, this does not appear to result from better noncovalent interactions with the enzyme; instead, improved affinity may be derived from significantly better entropic contributions to binding displayed by the phenethyl-substituted imidazole compound.

Probing Electrostatic Interactions along the Reaction Pathway of a Glycoside Hydrolase: Histidine Characterization by NMR Spectroscopy

“…Furthermore, this indicates that the pK a of the bound xylobio-imidazole is likely elevated from its solution value of 5.9 (J. Wicki and S. G. Withers, unpublished observations) due to favorable active site electrostatic interactions. This conclusion is also consistent with the atomic-resolution X-ray crystallographic structure of cationic glucoimidazole bound to a family 5 endoglucanase at pH 5.5 (48), and as inferred from the pH-dependent inhibition of a family 1 -glycosidase by glucoimidazole derivatives (49).…”

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

“…34 Most likely, the increased potency associated with the presence of the phenyl carbamate groups stems from desolvation of this substituent upon binding to NagZ, a phenomenon described for other glycoside hydrolases. 35 Together, these findings reveal that the NagZ active site accommodates inhibitors having bulky N-acyl moieties while retaining high binding affinity. These structures do not, however, offer insight into the basis of diminished binding of such inhibitors to GH20 and GH84 enzymes.…”

β-Glucosidases

“…The glucose of the incoming substrate has sometimes been observed to be distorted into a 1 S 3 skew boat as it moves toward the 4 H 3 half-chair shape in the first transition state, although in other structures it is poorly defined by the electron density, apparently due to high mobility [108][109][110][111][112]. The structures of certain putative transition state mimics have also been solved in the active site and shown to have a structure close to the 4 H 3 half-chair, although others appeared to inhibit by mechanisms other than transition state mimicry [108,[113][114][115][116][117][118].…”

“…In the second, deglycosylation step, the catalytic base (the same carboxylate as the catalytic acid) extracts a proton from a water molecule, improving its nucleophilic power to attack at the anomeric carbon and displace the enzyme. Hydrolysis by either mechanism is equivalent in the organism, since mutarotation of the released glucose will lead to a racemic mixture of glucose in solution after a short time rather similar and broad substrate specificities, the complexes of b-glycosidase S from the archaeon Sulfolobus solfataricus and b-glucosidase A from the eubacteria Thermotoga maritima with a range of inhibitors has provided a wealth of information on catalytic and inhibitory mechanisms [113][114][115][116][117][118]. In addition, site-directed mutagenesis of GH1 enzymes with and without experimentally determined structures has been done to test the roles of various residues.…”