PMID:2659075
| Citation |
Konopka, JM, Halkides, CJ, Vanhooke, JL, Gorenstein, DG and Frey, PA (1989) UDP-galactose 4-epimerase. Phosphorus-31 nuclear magnetic resonance analysis of NAD+ and NADH bound at the active site. Biochemistry 28:2645-54 |
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| 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. |
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| Keywords |
Binding Sites; Carbohydrate Epimerases/metabolism; Escherichia coli/enzymology; Magnetic Resonance Spectroscopy/methods; NAD/metabolism; Oxidation-Reduction; Phosphorus; Protein Binding; Protein Conformation; UDPglucose 4-Epimerase/antagonists & inhibitors; UDPglucose 4-Epimerase/metabolism; Uridine Monophosphate/pharmacology |
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