title: Biochemical and structural assessment of the 1-N-azasugar GalNAc-isofagomine as a potent family 20 beta-N-acetylhexosaminidase inhibitor.
abstract: Azasugar inhibitors of the isofagomine class are potent competitive inhibitors of configuration-retaining beta-glycosidases. This potency results from the formation of a strong electrostatic interaction between a protonated endocyclic nitrogen at the "anomeric" center of the inhibitor and the catalytic nucleophile of the enzyme. Although the majority of retaining beta-glycosidases use a mechanism involving a carboxylate residue as a nucleophile, Streptomyces plicatus beta-N-acetylhexos-aminidase (SpHEX) and related family 20 glycosidases lack such a catalytic residue and use instead the carbonyl oxygen of the 2-acetamido group of the substrate as a nucleophile to "attack" the anomeric center. Thus, a strong electrostatic interaction between the inhibitor and enzyme is not expected to occur; nonetheless, the 1-N-azasugar (2R,3R,4S,5R)-2-acetamido-3,4-dihydroxy-5-hydroxymethyl-piperidinium hydrochloride (GalNAc-isofagomine.HCl), which was synthesized and assayed for its ability to inhibit SpHEX, was found to be a potent competitive inhibitor of the enzyme (K(i) = 2.7 microm). A crystallographic complex of GalNAc-isofagomine bound to SpHEX was solved and refined to 1.75 A and revealed that the lack of a strong electrostatic interaction between the "anomeric" center of GalNAc-isofagomine and SpHEX is compensated for by a novel 2.8-A hydrogen bond formed between the equatorial proton of the endocyclic nitrogen of the azasugar ring and the carboxylate of the general acid-base residue Glu-314 of SpHEX. This interaction appears to contribute to the unexpected potency of GalNAc-isofagomine toward SpHEX.
authors.affiliation: Department of Biochemistry, Canadian Institutes of Heath Research Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: B L
authors.initials: BL
authors.lastname: Mark
authors.forename: D J
authors.initials: DJ
authors.lastname: Vocadlo
authors.forename: D
authors.initials: D
authors.lastname: Zhao
authors.forename: S
authors.initials: S
authors.lastname: Knapp
authors.forename: S G
authors.initials: SG
authors.lastname: Withers
authors.forename: M N
authors.initials: MN
authors.lastname: James
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006860:Hydrogen Bonding; D050112:Imino Pyranoses; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology*; D013302:Streptomyces / Q000201:enzymology*; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D050112:Imino Pyranoses; D010880:Piperidines; C098432:isofagomine; D001619:beta-N-Acetylhexosaminidases