Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic level

Abstract

Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate's structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (²H₃) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (²H₃) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.

Fig. 1

Select competitive inhibitors of glycoside hydrolases. a Representative natural products. b Natural product (Neu2en5Ac) inspired therapeutic agent (oseltamivir)

Fig. 2

Glycoside hydrolase catalysis and chemical structures for select inhibitors. a Mechanism for glucosylation with a natural substrate analog. b Proposed mechanism of pseudo-glucosylation for the allylic α-glucoside mimic 1. c Structures of mechanism-based 4-nitrophenyl covalent inhibitor 1; 2,4-dinitrophenyl β-glucoside analog 2; the α-galactose analog inhibitors 3a, b; the 2-deoxy-2-fluoro covalent inhibitors 4; 5, and 6 the hydrolyzed products for the α-galactose analog inhibitors 3 and 4, respectively; and two conventional glycoside hydrolase inactivators: 2-deoxy-2-fluorogalactoside (7) and cyclophellitol analog (8). For clarity, most hydroxyl groups are not shown for the transition state or intermediate in a, b

Fig. 3

Synthesis of the cyclohexene carbagalactose analog 3b

Fig. 4

Synthesis of the 2-deoxy-2-fluorocarbagalactose analog 4

Fig. 5

Structure of TmGalA in complex with 3a and 5. a Structure of TmGalA mutant D387A in complex with intact 3a. b Structure of TmGalA in complex with hydrolyzed inhibitor 5. The maximum likelihood/σ_A weighted 2F_obs−F_calc electron density map is contoured at 1.2 sigma in a and 2 sigma in b. The catalytic residues D327 and D387 (or D387A) residues are shown. c, d Structure of TmGalA in complex with hydrolyzed inhibitor 5 illustrating active site residues that hydrogen bond with the inhibitor. The red sphere in c and gray sphere in d represent a water molecule

Fig. 6

Structure of TmGalA in complex with 4. a Structure of TmGalA mutant D387A in complex with intact 4. b Structure of TmGalA in complex with 2-deoxy-2-fluorocarbagalactose fragment of 4 covalently bound to the nucleophile D327. c Structure of TmGalA in complex with hydrolyzed inhibitor 6. The maximum likelihood/σ_A weighted 2F_obs−F_calc electron density map is contoured at 1.5 sigma in all cases. The catalytic residues D327 and D387 (or D387A) residues are shown

Fig. 7

Structural snapshots of the active site of TmGalA. The enzyme's active site is shown in complex with: a 4 (or E:I); b 2-deoxy-2-fluorocarbagalactose fragment of 4 covalently bound to the nucleophile Asp327 (or E-I) and c hydrolyzed inhibitor 6 (or E:P). Structures were optimized at DFT/MM level of theory

Fig. 8

Scheme for the covalent inhibition of α-glycosidases by cyclohexene carbasugar analogs. a Mechanistic scheme showing individual rate constants. b Kinetic expressions for hydrolysis of inhibitor. c Kinetic expressions for the covalent labeling stopped-flow experiments

Fig. 9

Superposition of TmGalA structures. a Structure of TmGalA mutant D387A in complex with intact 4 (cyan) and of wild-type TmGalA in complex with 2-deoxy-2-fluorocarbagalactose fragment of 4 covalently bound to the nucleophile Asp327 (yellow). b Structure of wild-type TmGalA in complex with hydrolyzed inhibitor 6 (pink) and of wild-type TmGalA in complex with 2-deoxy-2-fluorocarbagalactose fragment of 4 covalently bound to the nucleophile Asp327 (yellow). c Structure of TmGalA mutant D387A in complex with intact 4 (cyan) and of wild-type TmGalA in complex with hydrolyzed inhibitor 6 (pink)