Inhibition of CMP-sialic acid transport into Golgi vesicles by nucleoside monophosphates.
We examined the interactions of nucleotides with the CMP-sialic acid transporter in order to better understand which features play a role in binding and to investigate the relationship between binding and subsequent transport. With respect to the sugar, the transporter requires a complete ribose ring for tight binding, and the 2'-ara hydrogen makes an important contact. The enzyme exhibits little specificity with respect to the 2'- and 3'-hydroxyls, as it tolerated substitutions ranging from fluorine to an azido group. In the base, the C4 amine and C2 carbonyl groups make important contacts, while the N3 nitrogen does not. However, adding a methyl group to N3 dramatically reduced binding, indicating that mass at this position sterically hinders binding. Adding a group at C5 had either no effect or slightly enhanced binding. To determine if the transporter recognizes these CMP analogues as substrates, we assayed them for their ability to trans stimulate CMP-sialic acid import. These data suggest that the enzyme transports a wide variety of NMPs, and the rate of transport is inversely proportional to the K(I) of the analogue. The importance of our findings for understanding the specificities of the different nucleotide-sugar tranlocators and the design of novel glycosylation inhibitors are discussed.
Chiaramonte
M
M
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Koviach
J L
JL
Moore
C
C
Iyer
V V
VV
Wagner
C R
CR
Halcomb
R L
RL
Miller
W
W
Melançon
P
P
Kuchta
R D
RD
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003568:Cytidine Monophosphate / Q000032:analysis; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry; D006031:Glycosylation / Q000187:drug effects; D006056:Golgi Apparatus / Q000378:metabolism*; D008099:Liver; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008956:Models, Chemical; D009711:Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D009712:Nucleotides, Cyclic / Q000032:analysis; D051381:Rats; D014542:Uridine Monophosphate / Q000032:analysis
D002352:Carrier Proteins; D008565:Membrane Proteins; D009711:Nucleotides; D009712:Nucleotides, Cyclic; C019509:nucleoside monophosphate sugars; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D014542:Uridine Monophosphate; D003568:Cytidine Monophosphate