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J Biol Chem. 1994 Dec 16;269(50):31496-504.

Characterization and activation of naturally occurring abortive complexes of UDP-galactose 4-epimerase from Escherichia coli.

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  • 1Institute for Enzyme Research, Graduate School.


UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose. The enzyme from Escherichia coli is a dimeric protein with an overall molecular weight of 79,000 that contains NAD+ very tightly but noncovalently bound in the enzymatic active site. NAD+ is the coenzyme for epimerization and is transiently reduced to NADH in the course of catalysis. All samples of highly purified UDP-galactose 4-epimerase contain significant amounts of NADH, and that purified after overexpression in E. coli cells contains a substantial amount of NADH. To the degree that NADH replaces enzyme bound NAD+ in the coenzyme binding site, the epimerase activity is decreased. The extinction coefficient at 345 nm for NADH in its binding site is estimated to be 3.3 mM-1 cm-1. 31P NMR spectroscopic and enzymatic analyses reveal that UDP-glucose, UDP-galactose, UDP, and UMP are gradually released from the purified enzyme upon addition of UMP or P1-5'-uridine-P2-methyl diphosphate (MeUDP). It is concluded that NADH associated with the purified enzyme is a component of inactive, abortive complexes (E-NADH-uridine nucleotide) that contain tightly bound uridine nucleotides in place of the epimerization intermediate UDP-4-keto-alpha-D-hexoglucopyranose. These complexes are produced in vivo in the course of bacterial growth. The enzymatic activity of purified epimerase is increased by reaction with 1,2-naphthoquinone-4-sulfonate, which oxidizes the NADH to NAD+. Compositionally defined abortive complexes (E-NADH-uridine nucleotide) containing UMP, UDP, or UDP-hexoses (Glc/Gal) have been prepared in vitro and subjected to activation by 1,2-naphthoquinone-4-sulfonate. All are activated at rates comparable to that for the purified enzyme, although those containing UMP and UDP-hexose are more readily activated than those containing UDP. The activity of the reactivated enzyme approaches that of the most highly active epimerase that has been reported from E. coli.

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