title: UDP-galactose 4-epimerase. Phosphorus-31 nuclear magnetic resonance analysis of NAD+ and NADH bound at the active site.
abstract: The phosphorus atoms of NAD+ bound within the active site of UDP-galactose 4-epimerase from Escherichia coli exhibit two NMR signals, one at delta = -9.60 +/- 0.05 ppm and one at delta = -12.15 +/- 0.01 ppm (mean +/- standard deviation of four experiments) relative to 85% H3PO4 as an external standard. Titration of epimerase.NAD+ with UMP causes a UMP-dependent alteration in the chemical shifts of the resulting exchange-averaged spectra, which extrapolate to delta = -10.51 ppm and delta = -11.06 ppm, respectively, for the fully liganded enzyme, with an interconversion rate between epimerase.NAD+ and epimerase.NAD+.UMP of at least 490 s-1. Conversely, the binding of 8-anilinonaphthalene-1-sulfonate, which is competitive with UMP, causes a significant sharpening of the epimerase.NAD+ resonances but very little alteration in their chemical shifts, to delta = -9.38 ppm and delta = -12.16 ppm, respectively. UMP-dependent reductive inactivation by glucose results in the convergence of the two resonances into a single signal of delta = -10.57 ppm, with an off-rate constant for UMP dissociation from the epimerase.NADH.UMP complex estimated at 8 s-1. Reductive inactivation by borohydride under anaerobic conditions yields a single, broad resonance centered at about delta = -10.2 ppm. The data are consistent with, and may reflect, the activation of NAD+ via a protein conformational change, which is known from chemical studies to be driven by uridine nucleotide binding. Incubation of epimerase.NAD+ with UMP in the absence of additional reducing agents causes a very slow reductive inactivation of the enzyme with an apparent pseudo-first-order rate constant of 0.013 +/- 0.001 h-1, which appears to be associated with liberation of inorganic phosphate from UMP.
authors.affiliation: Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705.
authors.forename: J M
authors.initials: JM
authors.lastname: Konopka
authors.forename: C J
authors.initials: CJ
authors.lastname: Halkides
authors.forename: J L
authors.initials: JL
authors.lastname: Vanhooke
authors.forename: D G
authors.initials: DG
authors.lastname: Gorenstein
authors.forename: P A
authors.initials: PA
authors.lastname: Frey
mesh_terms: D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010758:Phosphorus; D011485:Protein Binding; D011487:Protein Conformation; D014534:UDPglucose 4-Epimerase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000494:pharmacology
chemical_list: D009243:NAD; D010758:Phosphorus; D014542:Uridine Monophosphate; D002238:Carbohydrate Epimerases; D014534:UDPglucose 4-Epimerase