Bidirectional N-acetylglucosamine transfer mediated by beta-1,4-N-acetylglucosaminyltransferase III.
beta-1,4-N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of the bisecting GlcNAc and plays a regulatory role in the biosynthesis of the N-linked oligosaccharide. In this study, we examined whether the glycosyl transfer catalyzed by GnT-III is reversible, and, in addition, investigated the equilibrium of the GnT-III-catalyzed reaction. Incubation of the agalactosyl-bisected biantennary oligosaccharide with GnT-III in the presence of the sufficiently high concentration of uridine diphosphate (UDP) resulted in conversion of the bisected oligosaccharide into the nonbisected one. This reaction was accompanied by the stoichiometric formation of UDP-GlcNAc, which appeared to result from the transfer of GlcNAc from the oligosaccharide to UDP. Thus, these results indicate that GnT-III is capable of perceivably catalyzing the reverse reaction in vitro, as found in some glycosyltransferases. When the equilibrium of the reaction was kinetically analyzed, it was found that the state of the equilibrium is greatly displaced toward the formation of the bisecting GlcNAc. In terms of free energy change, as estimated, the reaction by GnT-III can be comparable to the hydrolysis of ATP. Although GnT-III catalyzes bidirectional transfer of GlcNAc between the oligosaccharide and UDP, the removal of the bisecting GlcNAc is unlikely in vivo, due to the displacement of the equilibrium. It is known that equilibria of certain glycosyltransferase reactions are not biased as greatly as the case of GnT-III, and thus it seems likely that there are a variety of equilibrium states in glycosyltransferase reactions. In living cells, the assembly of oligosaccharides could be regulated by not only rate control but also equilibrium control.
Okada
Takahiro
T
Division of Molecular Cell Biology, Department of Biomolecular Sciences, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan.
Ihara
Hideyuki
H
Ito
Ritsu
R
Taniguchi
Naoyuki
N
Ikeda
Yoshitaka
Y
D000117:Acetylglucosamine / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014530:Uridine Diphosphate / Q000737:chemistry* / Q000378:metabolism
D009844:Oligosaccharides; D011994:Recombinant Proteins; D014530:Uridine Diphosphate; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine