Uridine diphosphate galactose-4-epimerase. Uridine monophosphate-dependent reduction by alpha- and beta-D-glucose.

U G Kang,L D Nolan,P A Frey

doi:10.1016/s0021-9258(19)40914-9

Abstract

Rates of UMP-dependent reduction of the DPN+ associated with Escherichia coli UDP-galactose-4-epimerase at 27 degrees and 0.2 M ionic strength in 0.1 M Tris-HCl buffer, pH 8.5, are reported. The reaction exhibits excellent pseudo-first order behavior when D-glucose is at anomeric equilibrium. The effects of [UMP] and [glucose] on the observed first order rate constants are consistent with the following equation. The symbols phi are empirical parameters. (See article). The data indicate that the pathway involves random equilibrium binding of UMP and glucose followed by rate-limiting decomposition of the ternary complex to epimerase-DNPH. The binding parameters indicate that the principal activating effect of UMP is not simply to increase the affinity of the enzyme for glucose. UMP appears to increase the reactivity or availability of enzyme-bound DPN+. The kinetic isotope effect for the reaction of D-]1-2H]glucose (kH/kD) is 4.2, which confirms that C-1 is oxidized and that hydride transfer is rate limiting. Both of the purified anomers, alpha- and beta-D-glucose, reduce the enzyme-bound DPN+. As indicated by the deviations from pseudo-first order kinetics because of concurrent mutarotation, the beta anomer is the more reactive, reacting about 4 to 5 times faster than the alpha anomer at concentrations well below saturation. Is is suggested that the lack of stereo-specificity in this reaction may be attributed to the two anomers being productively bound with their opposite faces projecting toward C-4 of bound DPN+. Nonstereospecific oxidation of alpha- and beta-D-glucose may be a model for the mechanism of UDP-hexose epimerization, which also involves nonstereospecific hydride transfer.

Highlights

Rates of UMP-dependent reduction of the DPN+ associated with Escherichia co2i UDP-galactose-4-epimeraseat 27” and 0.2 M ionic strength in 0.1 M Tris .HCl buffer, pH 8.5, are reported
The reaction exhibits excellent pseudo-first order behavior when D-glucose is at anomeric equilibrium
The effects of [UMP] and [glucose] on the observed first order rate constants are consistent with the following equation

Summary

Methods

Enzyme-We purified and assayed UDP-galactose-4-epimerase by the published procedure We made one modification in the procedure. We carried out the hydroxylapatite chromatography step at pH 7.2 rather than at pH 6.5. The column equilibration, charging, and elution were exactly as described (lo), except that all buffers were at pH 7.2. The enzyme would not be eluted from the column with the gradient described, but at pH 7 to 7.2, the elution results were equivalent to the published data [10] We found that at pH 6.5. the enzyme would not be eluted from the column with the gradient described, but at pH 7 to 7.2, the elution results were equivalent to the published data [10]

Results

Discussion

Conclusion