Mechanism of Human α-1,3-Fucosyltransferase V:  Glycosidic Cleavage Occurs Prior to Nucleophilic Attack

Abstract

R-1,3-Fucosyltransferase V (FucT V) catalyzes the transfer of l-fucose from the donor sugar guanosine 5′-diphospho--l-fucose (GDP-Fuc) to an acceptor sugar. A secondary isotope effect on the fucosyltransfer reaction with guanosine 5′-diphospho-[1-2 H]--l-fucose (GDP-[1-2 H]-Fuc) as the substrate was observed and determined to be D V ) 1.32 ( 0.13 and D V/K ) 1.27 ( 0.07. Competitive inhibition of FucT V by guanosine 5′-diphospho-2-deoxy-2-fluoro--l-fucose (GDP-2F-Fuc) was observed with an inhibition constant of 4.2 µM which represents the most potent inhibitor of this enzyme to date. Incubation of GDP-2F-Fuc with FucT V and an acceptor molecule prior to the addition of GDP-Fuc had no effect on the potency of inhibition, indicating that GDP-2F-Fuc is neither an inactivator nor a slow substrate. Both the observed secondary isotope effect and the inhibition by GDP-2F-Fuc are consistent with a charged, sp 2 -hybridized, transition-state structure. A convenient and efficient synthesis of GDP-[1-2 H]-Fuc and GDP-2F-Fuc and a nonradioactive, fluorescence assay for fucosyltransferase activity have been developed.The R-1,3-fucosylated oligosaccharide structures are central to numerous cell-cell interactions (Ichikawa et al., 1994) such as inflammation, tumor development, and blood clotting (Foxall et al., 1992;Parekh & Edge, 1994). Five distinct human R-1,3-fucosyltransferases have been cloned (KukowskaLatallo et al., 1990;Lowe et al., 1991;McCurley et al., 1995;Reguigne-Arnould et al., 1995;Sasaki et al., 1994; Weston et al., 1992a,b) and shown to have different acceptor sugar specificity. R-1,3-Fucosyltransferase V (FucT V) 1 is responsible for the terminal step in the biosynthesis of Lewis x (Le x ) and sialyl Lewis x (sLe x ) (Figure 1), a tetrasaccharide ligand involved in inflammatory cell adhesion and metastasis (Lasky, 1992;Muramatsu, 1993). Thus inhibition of this enzyme may impede inflammation or cancer progression, and understanding the mechanism of FucT V may lead to the development of effective inhibitors.The R-1,3-fucosyltransferase V-catalyzed reaction proceeds with inversion of configuration at the anomeric center of l-fucose (Weston et al., 1992a). Product inhibition studies have been used to establish that FucT V has an ordered, sequential, bi-bi mechanism with guanosine 5′-diphospho--l-fucose (GDP-Fuc) binding first and the product GDP releasing last (Qiao et al., 1996). FucT V has been shown to have a catalytic residue with pK a ) 4.1, presumably an active-site carboxylate residue . A solvent isotope effect was observed (D V ) 2.9, D V/K ) 2.1) and exploited in a proton inventory study to show that there is a one-proton transfer in the transition state . The transition-state structure of glycosyltransferasecatalyzed reactions has been proposed to have a flattened half-chair conformation with substantial oxocarbenium ion character at the anomeric position (Kim et al., 1988;Murray et al., 1996), analogous to that of the glycosidase reactions (Look et al., 1993;Sinnott, 1990). Consistent with...

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

“…The involvement of an oxocarbenium ion transition state in the chemical mechanisms of glycosyltransferases and glycosidases is well supported. Significant normal ␣-deuterium secondary isotope effects have been reported for inverting and retaining glycosyltransferases (45)(46)(47) and glycosidases (48,49) consistent with C1-O bond cleavage occurring prior to attack by the acceptor sugar or water. For inverting glycosidases and glycosyltransferases, the proposed single displacement mechanism is well established (50).…”

C-terminal Amino Acids of Helicobacter pylori α1,3/4 Fucosyltransferases Determine Type I and Type II Transfer

“…Mammalian ␣1,3/4 FucTs, especially human FucTs, have been extensively characterized with regard to the key polar group of the acceptors (25, 29), the domain or amino acid residues responsible for donor binding (30 -33) and acceptor recognition (15-18, 29 -32), and kinetic mechanisms (33)(34)(35). The study of bacterial ␣1,3/4 FucTs, in contrast, is not as far advanced.…”

A Single Amino Acid in the Hypervariable Stem Domain of Vertebrate α1,3/1,4-Fucosyltransferases Determines the Type 1/Type 2 Transfer

“…Therefore, these His, Glu, and Asp residues can be considered as candidates for the formation of hydrogen bonds with the acceptor oligosaccharide. Based on recent studies, it seems reasonable to suggest that the active-site base in the protein may be a carboxylate anion (33,34). In another study, Britten and Bird (35) investigated the amino acids essential for the activity of Fuc-TVI through chemical modification, and they concluded that the substratebinding site of the enzyme possesses His residue(s) that are essential for enzyme activity.…”