Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases.
The glycosyltransferases controlling the biosynthesis of cell-surface complex carbohydrates transfer glycosyl residues from sugar nucleotides to specific hydroxyl groups of acceptor oligosaccharides. These enzymes represent prime targets for the design of glycosylation inhibitors with the potential to specifically alter the structures of cell-surface glycoconjugates. With the aim of producing such inhibitors, synthetic oligosaccharide substrates were prepared for eight different glycosyltransferases. The enzymes investigated were: A, alpha(1----2, porcine submaxillary gland); B, alpha(1----3/4, Lewis); C, alpha(1----4, mung bean); D, alpha(1----3, Lex)-fucosyltransferases; E, beta(1----4)-galactosyltransferase; F, beta(1----6)-N-acetylglucosaminyltransferase V; G, beta(1----6)-mucin-N-acetylglucosaminyltransferase ("core-2" transferase); and H, alpha(2----3)-sialyltransferase from rat liver. These enzymes all transfer sugar residues from their respective sugar nucleotides (GDP-Fuc, UDP-Gal, UDP-GlcNAc, and CMP-sialic acid) with inversion of configuration at their anomeric centers. The Km values for their synthetic oligosaccharide acceptors were in the range of 0.036-1.3 mM. For each of these eight enzymes, acceptor analogs were next prepared where the hydroxyl group undergoing glycosylation was chemically removed and replaced by hydrogen. The resulting deoxygenated acceptor analogs can no longer be substrates for the corresponding glycosyltransferases and, if still bound by the enzymes, should act as competitive inhibitors. In only four of the eight cases examined (enzymes A, C, F, and G) did the deoxygenated acceptor analogs inhibit their target enzymes, and their Ki values (all competitive) remained in the general range of the corresponding acceptor Km values. No inhibition was observed for the remaining four enzymes even at high concentrations of deoxygenated acceptor analog. For these latter enzymes it is suggested that the reactive acceptor hydroxyl groups are involved in a critical hydrogen bond donor interaction with a basic group on the enzyme which removes the developing proton during the glycosyl transfer reaction. Such groups are proposed to represent logical targets for irreversible covalent inactivation of this class of enzyme.
Hindsgaul
O
O
Department of Chemistry, University of Alberta, Edmonton, Canada.
Kaur
K J
KJ
Srivastava
G
G
Blaszczyk-Thurin
M
M
Crawley
S C
SC
Heerze
L D
LD
Palcic
M M
MM
D000818:Animals; D002417:Cattle; D005647:Fucosyltransferases / Q000378:metabolism; D006602:Hexosyltransferases / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D013363:Submandibular Gland / Q000201:enzymology; D013379:Substrate Specificity; D013552:Swine
D009844:Oligosaccharides; D005647:Fucosyltransferases; D006602:Hexosyltransferases; D010100:Oxygen