Potent inhibition of a GH20 exo-β-N-acetylglucosaminidase from marine Vibrio bacteria by reaction intermediate analogues

Exo-β-N-acetylglucosaminidases (GlcNAcases) are hydrolytic enzymes involved in the metabolism of chitin in bacteria and in eukaryotic glycosphingolipid metabolism, with genetic defects in human GlcNAcases (HexA and HexB) resulting in Tay-Sachs and Sandhoff diseases, respectively. Here, we determined the effects of three known inhibitors of exo-β-N-acetylglucosaminidases (PUGNAc, NHAcCAS and NHAcDNJ) on a GH20 exo-β-N-GlcNAcase (VhGlcNAcase) from the pathogenic bacterium Vibrio harveyi, in dose-response experiments. The inhibitors were shown to modify the kinetic parameters (both K_m and k_cat), yielding significant decreases in the overall efficiency of the enzyme in hydrolyzing the natural substrate diNAG. Molecular interactions between the inhibitors and the enzyme were investigated by isothermal calorimetry (ITC), and were confirmed using molecular docking. VhGlcNAcase was strongly inhibited by these compounds, with PUGNAc having the lowest IC₅₀ value, of 1.2 μM. Molecular docking suggested that the inhibitors mimicked reaction intermediates, with enzyme-inhibitor interactions being similar to those of the enzyme with diNAG. The equilibrium dissociation constants (K_d) obtained from ITC were 0.19 μM for PUGNAc, 12.9 μM for NHAcCAS and 25.6 μM for NHAcDNJ, confirming that PUGNAc was the most potent inhibitor. The ITC data indicated that the binding of the enzyme to the inhibitors was driven by enthalpy. The negative heat capacity change (ΔC_p) of −0.34 ± 0.05 kcal·mol⁻¹·K⁻¹ indicates that hydrophobic interactions make a substantial contribution to the molecular interactions between PUGNAc and the enzyme. Our results suggest that PUGNAc is a highly potent inhibitor, and suggest its usefulness as a scaffold for potential drugs targeting GlcNAcase-related metabolic diseases.