UDP-galactose 4-epimerase. Phosphorus-31 nuclear magnetic resonance analysis of NAD+ and NADH bound at the active site

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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 .cntdot. 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 .cntdot. NAD+ and epimerase .cntdot. NAD+ .cntdot. 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 .cntdot. 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 .cntdot. NADH .cntdot. 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 .cntdot. 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.