title: Using substrate analogues to probe the kinetic mechanism and active site of Escherichia coli MenD.
abstract: MenD catalyzes the thiamin diphosphate-dependent decarboxylative carboligation of α-ketoglutarate and isochorismate. The enzyme is essential for menaquinone biosynthesis in many bacteria and has been proposed to be an antibiotic target. The kinetic mechanism of this enzyme has not previously been demonstrated because of the limitations of the UV-based kinetic assay. We have reported the synthesis of an isochorismate analogue that acts as a substrate for MenD. The apparent weaker binding of this analogue is advantageous in that it allows accurate kinetic experiments at substrate concentrations near K(m). Using this substrate in concert with the dead-end inhibitor methyl succinylphosphonate, an analogue of α-ketoglutarate, we show that MenD follows a ping-pong kinetic mechanism. Using both the natural and synthetic substrates, we have measured the effects of 12 mutations of residues at the active site. The results give experimental support to previous models and hypotheses and allow observations unavailable using only the natural substrate.
authors.affiliation: Department of Chemistry, University of Saskatchewan, Saskatoon, SK S7N 5C9, Canada.
authors.forename: Maohai
authors.initials: M
authors.lastname: Fang
authors.forename: Andrea
authors.initials: A
authors.lastname: Macova
authors.forename: Kimberly L
authors.initials: KL
authors.lastname: Hanson
authors.forename: Jillian
authors.initials: J
authors.lastname: Kos
authors.forename: David R J
authors.initials: DR
authors.lastname: Palmer
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002827:Chorismic Acid / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011771:Pyruvate Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; C052985:isochorismic acid; C534108:2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase, E coli; D011771:Pyruvate Oxidase; D002827:Chorismic Acid