title: A mechanism-based inactivator of glycoside hydrolases involving formation of a transient non-classical carbocation.
abstract: The design of mechanism-based enzyme inactivators to generate chemical probes for biological research is an important challenge in carbohydrate chemistry. Here we describe the synthesis and biological evaluation of a novel carbocyclic mechanism-based inactivator of galactosidases (glycoside hydrolase families 27 and 36). Upon catalysis of this unnatural substrate, a transient non-classical carbocation forms within the enzyme active site. We show that the inactivation event, which proceeds via a bicyclobutonium ion intermediate, leads to a single alkylation event that occurs on the enzymatic nucleophile, an aspartic acid residue in this case. We also show that the catalytic proficiencies for enzymatic hydrolysis of substrates and inactivation by our bicyclo[4.1.0]heptyl analogue of galactose differ by only a factor of 20. This inactivator has the potential for further development as a useful biological research tool for both basic research and biotechnological applications.
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Saswati
authors.initials: S
authors.lastname: Chakladar
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Thomas
authors.initials: T
authors.lastname: Clark
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Lydia
authors.initials: L
authors.lastname: Cheng
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Shirley
authors.initials: S
authors.lastname: Ko
authors.affiliation: 1] Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 [2] Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bennet
mesh_terms: D000478:Alkylation; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D005690:Galactose / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry*; D005696:Galactosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D020124:Thermotoga maritima / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005696:Galactosidases; D005690:Galactose