Uridine diphosphate galactose 4-epimerase. Alkylation of enzyme-bound diphosphopyridine nucleotide by p-(bromoacetamido)phenyl uridyl pyrophosphate, an active-site-directed irreversible inhibitor.

When UDP-galactose 4-epimerase is inactivated by p-(bromoacetamido)phenyl uridyl pyrophosphate (BUP), the diphosphopyridine nucleotide (DPN) associated with this enzyme as a tightly bound coenzyme cannot be reduced by substrates or by UMP-activated reduction by glucose. Upon acid denaturation of the inactivated enzyme, the DPN released corresponded to 15-30% of that released from the native enzyme. When the enzyme is inactivated by [14C]BUP, about 80% of the radioactivity bound at the active site is released from the protein upon acid denaturation. When epimerase-[3H]DPN is inactivated with [14C]BUP, the 3H and 14C released from the protein upon denaturation of the complex cochromatograph on DEAE-Sephadex. Experiments with [nicotinamide-4-3H]DPN and [adenine-2,8-3H]DPN show that it is the adenine ring that is alkylated. The data suggest that the adenine ring of DPN in epimerase-DPN may be oriented near the glycosyl-binding subsite of this enzyme. Since the nicotinamide ring must also be near this site, it appears that the DPN may not be in an extended conformation when it is bound at the active site of UDP-galactose 4-epimerase from Escherichia coli.