Glycosyltransferase mechanisms: impact of a 5-fluoro substituent in acceptor and donor substrates on catalysis

Abstract

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.

Figure 1

Proposed weakly associative transition state for glycosyltransferases involving a UDP-GlcNAc donor and various GlcNAc-containing acceptor substrates.

Figure 2

Inhibition of CLS by UDP-(5-F)-GlcNAc. Each assay was carried out for 5 min as described in Materials and Methods using partially purified CLS (3.6 μg), UDP-(5-F)-GlcNAc (0.25 mM or 1 mM), and UDP-GlcNAc (165 μM-1.5 mM).

Figure 3

Double reciprocal plots of GalT-catalyzed glycosylation of GlcNAc-β-octyl glycoside (3A, 3B) and (5-F)-GlcNAc-β-octyl glycoside (3C, 3D). The data were fit to eq. 1 (see text for details), and the lines shown are derived from inserting the fitted parameters into eq. 1.

Scheme 1

Scheme 2

Scheme 3