title: Active site mutants of the "non-hydrolyzing" UDP-N-acetylglucosamine 2-epimerase from Escherichia coli.
abstract: The "non-hydrolyzing" bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This homodimeric enzyme is allosterically activated by its substrate, UDP-GlcNAc, and it is thought that one subunit plays a regulatory role, while that of the other plays a catalytic role. In this work, five active site mutants were prepared (D95N, E117Q, E131Q, K15A, and H213N) and analyzed in terms of their effects on binding, catalysis, and allosteric regulation. His213 appears to play a role in UDP binding and may also assist in catalysis and/or regulation, but is not a key catalytic residue. Lys15 appears to be quite important for binding. All three of the carboxylate mutants showed dramatic decreases in the value of k(cat) but relatively unaffected values of K(M). Thus, these residues are playing key roles in catalysis and/or regulation. In the case of E117Q, the reaction intermediates are released into solution at a rate comparable to that of the overall catalysis. This may indicate that Glu117 plays the role as an acid/base catalyst in the second step of the UDP-GlcNAc epimerization reaction. All three carboxylate mutants were found to exhibit impaired allosteric control.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Jomy
authors.initials: J
authors.lastname: Samuel
authors.forename: Martin E
authors.initials: ME
authors.lastname: Tanner
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D014530:Uridine Diphosphate / Q000737:chemistry / Q000378:metabolism
chemical_list: D014530:Uridine Diphosphate; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase