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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.

Bacteria often produce toxins which kill competing bacteria. Colicins, produced by and toxic to Escherichia coli bacteria are three-domain proteins so efficient that one molecule can kill a cell. The C-terminal domain carries the lethal activity and the central domain is required for surface receptor binding. The N-terminal domain, required for translocation across the outer membrane, is always intrinsically unstructured. It has always been assumed therefore that the C-terminal cytotoxic domain is required for the bactericidal activity. Here we report the unexpected finding that in isolation, the 90-residue unstructured N-terminal domain of colicin N is cytotoxic. Furthermore it causes ion leakage from cells but, unlike known antimicrobial peptides (AMPs) with this property, shows no membrane binding behaviour. Finally, its activity remains strictly dependent upon the same receptor proteins (OmpF and TolA) used by full-length colicin N. This mechanism of rapid membrane disruption, via receptor mediated binding of a soluble peptide, may reveal a new target for the development of highly specific antibacterials.

Glycoside hydrolases (GHs) have attracted considerable attention as targets for therapeutic agents, and thus mechanism‐based inhibitors are of great interest. We report the first structural analysis of a carbocyclic mechanism‐based GH inactivator, the results of which show that the two Michaelis complexes are in 2H3 conformations. We also report the synthesis and reactivity of a fluorinated analogue and the structure of its covalently linked intermediate (flattened 2H3 half‐chair). We conclude that these inactivator reactions mainly involve motion of the pseudo‐anomeric carbon atom, knowledge that should stimulate the design of new transition‐state analogues for use as chemical biology tools.

Glycoside hydrolases (GHs) have attracted considerable attention as targets for therapeutic agents, and thus mechanism-based inhibitors are of great interest. We report the first structural analysis of a carbocyclic mechanism-based GH inactivator, the results of which show that the two Michaelis complexes are in 2 H 3 conformations. We also report the synthesis and reactivity of a fluorinated analogue and the structure of its covalently linked intermediate (flattened 2 H 3 half-chair). We conclude that these inactivator reactions mainly involve motion of the pseudo-anomeric carbon atom, knowledge that should stimulate the design of new transition-state analogues for use as chemical biology tools. Lifeissupportedbyamyriadofenzyme-catalyzedreactions;one such life-sustaining activity is the transfer of carbohydrate groups from one biomolecule to another.[1] Understanding how these fundamentally important transfer reactions occur in nature guides researchers in the design of compounds (inhibitors/activators) that modulate the activity of these biological catalysts. Glycoside hydrolases (GHs or glycosidases) are a type of carbohydrate-processing enzyme used in the reshaping of biomolecules.[2] Most GHs catalyze glycoside hydrolysis through one of two distinct processes that are reliant on a pair of active-site aspartic and/or glutamic acid residues. Hydrolysis by such retaining glycosidases involves two sequential inversions of configuration at the anomeric center, the first of which results in the formation of a covalent glycosyl-enzyme intermediate (Figure 1 a). In contrast, inverting glycosidases operate via a single inversion of configuration at the anomeric center. In both cases, pyranosylium ion like transition states (TSs), which can have half-chair ( 4 H 3 / 3 H 4 ), boat ( 2,5 B/B 2,5 ), or envelope ( 4 E and 3 E) conformations (Figure 1 b), [2,3] are implicated. By exploiting this knowledge, we recently reported that the cyclopropyl-containing carbasugar 1 is a mechanism-based inactivator of an a-d-galactosidase from Thermotoga maritima (TmGalA; Figure 1 c). [4] Within the enzymatic active site, 1 likely forms a transient bicyclobutenium ion (1 + ), and enzyme inactivation occurs through alkylation of the catalytic nucleophile Asp 327.Given the current desire for small-molecule transitionstate analogues (TSAs) as leads for therapeutic development, [5] it is important to understand how GHs stabilize cationic TSs.[4] Of note, GHs are among the most catalytically proficient enzymes, since they accelerate hydrolysis of glycosidic bonds by up to 10 17 -fold.[6] Therefore, an understanding of the distinct ring conformations of the substrate