JBC, Vol. 250, Issue 18, 7099-7105, Sep, 1975
Uridine diphosphate galactose-4-epimerase. Uridine monophosphate-dependent reduction by alpha- and beta-D-glucose
U. G. Kang, L. D. Nolan and P. A. Frey
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.
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