Glycosyltransferase mechanisms: impact of a 5-fluoro substituent in acceptor and donor substrates on catalysis.
In glycosyltransferase-catalyzed reactions a new carbohydrate-carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide donor or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electron-withdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis. In particular, we have investigated the effects of the 5-fluoro analogues on the kinetics of two glycosyltransferase-catalyzed reactions mediated by UDP-GlcNAc:GlcNAc-P-P-Dol N-acetylglucosaminyltransferase (chitobiosyl-P-P-lipid synthase, CLS) and beta-N-acetylglucosaminyl-beta-1,4 galactosyltransferase (GalT). The 5-fluoro group has a marked effect on catalysis when inserted into the UDP-GlcNAc donor, with the UDP(5-F)-GlcNAc serving as a competitive inhibitor of CLS rather than a substrate. The (5-F)-GlcNAc beta-octyl glycoside acceptor, however, is an excellent substrate for GalT. Both of these results support a weakly associative transition state for glycosyltransferase-catalyzed reactions that proceed with inversion of configuration.
Hartman
Matthew C T
MC
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Jiang
Songmin
S
Rush
Jeffrey S
JS
Waechter
Charles J
CJ
Coward
James K
JK
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D016695:Glycosyltransferases / Q000378:metabolism*; D007553:Isotope Labeling; D007700:Kinetics; D013379:Substrate Specificity; D014316:Tritium; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry / Q000378:metabolism
D014316:Tritium; D014537:Uridine Diphosphate N-Acetylglucosamine; D016695:Glycosyltransferases; D000117:Acetylglucosamine