Synthesis of the acceptor analog alphaFuc(1-->2)alphaGal-O(CH2)7 CH3: a probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase.
We report the chemical synthesis of alphaFuc(1-->2)alphaGal-O(CH2)7CH3 (1) an analog of the natural blood group (O)H disaccharide alphaFuc(1-->2)betaGal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog (alphaGal(-->3)alphaFuc(1-->2)]alphaGal-O(CH2)7CH3+ ++ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide alphaFuc(1-->2)betaGal-O(CH2)7CH3 (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.
Kamath
V P
VP
Department of Chemistry, University of Alberta, Edmonton, Canada.
Seto
N O
NO
Compston
C A
CA
Hindsgaul
O
O
Palcic
M M
MM
D000017:ABO Blood-Group System; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002384:Catalysis; D004187:Disaccharides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D005700:Galactosyltransferases / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D000017:ABO Blood-Group System; D004187:Disaccharides; C413420:Fuc(1-2)Gal-O(CH2)7-CH3; D011994:Recombinant Proteins; D014312:Trisaccharides; D014531:Uridine Diphosphate Galactose; D005700:Galactosyltransferases; C023155:blood-group-substance alpha-D-galactosyltransferase