title: Nucleophilic participation of reduced flavin coenzyme in mechanism of UDP-galactopyranose mutase.
abstract: UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(β) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ρ = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, Department of Chemistry and Biochemistry and Institute of Cellular and Molecular Biology, University of Texas, Austin, Texas 78712-0128, USA.
authors.forename: He G
authors.initials: HG
authors.lastname: Sun
authors.forename: Mark W
authors.initials: MW
authors.lastname: Ruszczycky
authors.forename: Wei-Chen
authors.initials: WC
authors.lastname: Chang
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Thibodeaux
authors.forename: Hung-Wen
authors.initials: HW
authors.lastname: Liu
mesh_terms: D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics
chemical_list: D029968:Escherichia coli Proteins; D005182:Flavin-Adenine Dinucleotide; D019751:Intramolecular Transferases; C490824:Glf protein, E coli