Inhibition of CMP-sialic acid transport into Golgi vesicles by nucleoside monophosphates

We examined the interactions of nucleotides with the CMP-sialic acid transp orter in order to better understand which features play a role in binding a nd to investigate the relationship between binding and subsequent transport . With respect to the sugar, the transporter requires a complete ribose rin g for tight binding, and the 2'-ara hydrogen makes an important contact. Th e enzyme exhibits little specificity with respect to the 2'- and 3'-hydroxy ls, as it tolerated substitutions ranging from fluorine to an azido group. In the base, the C4 amine and C2 carbonyl groups make important contacts, w hile the N3 nitrogen does not. However, adding a methyl group to N3 dramati cally reduced binding, indicating that mass at this position sterically hin ders binding. Adding a group at C5 had either no effect or slightly enhance d binding. To determine if the transporter recognizes these CMP analogues a s substrates, we assayed them for their ability to trans stimulate CMP-sial ic acid import. These data suggest that the enzyme transports a wide variet y of NMPs, and the rate of transport is inversely proportional to the K-I o f the analogue. The importance of our findings for understanding the specif icities of the different nucleotide-sugar tranlocators and the design of no vel glycosylation inhibitors are discussed.