SYNTHETIC SUBSTRATE-ANALOGS FOR UDP-GLCNAC - MAN-ALPHA-1-3R BETA-1-2-N-ACETYLGLUCOSAMINYLTRANSFERASE .1. SUBSTRATE-SPECIFICITY AND INHIBITORS FOR THE ENZYME

UDP-GlcNAc:Man alpha 1-3R beta 1-2-N-acetylglucosaminyltransferase I ( GlcNAc-TI; EC 2.4.1.101) catalyses the conversion of [Man alpha 1-6(Ma n alpha 1-3)Man alpha 1-6][Man alpha 1-3]Man beta-O-R to [Man alpha 1- 6(Man alpha 1-3)Man alpha 1-6] [GlcNAc beta 1-2Man alpha 1-3]Man beta- O-R (R = 1-4GlcNAc beta 1-4GlcNAc-Asn-X) and thereby controls the conv ersion of oligomannose to complex and hybrid asparagine-linked glycans (N-glycans). GlcNAc-TI also catalyses the conversion of Man alpha 1-6 (Man alpha 1-3)Man beta-O-octyl to Man alpha 1-6(GlcNAc beta 1-2Man al pha 1-3)Man beta-O-octyl. We have therefore tested a series of synthet ic analogues of Man ''alpha 1-6(Man'alpha 1-3)Man beta-O-octyl as subs trates and inhibitors for rat liver GlcNAc-TI. The 2 ''-deoxy and the 3 ''-, 4 ''- and 6 ''-O-methyl derivatives are all good substrates con firming previous observations that the hydroxyl groups of the Man ''al pha 1-6 residue do not play major roles in the binding of substrate to enzyme. In contrast, all four hydroxyl groups on the Man'alpha 1-3 re sidue are essential since the corresponding deoxy derivatives either d o not bind (2'- and 3'-deoxy) or bind very poorly (4'- and 6'-deoxy) t o the enzyme. The 2'- and 3'-O-methyl derivatives also do not bind to the enzyme. However, the 4'-O-methyl derivative is a substrate (K-M = 2.6 mM) and the 6'-O-methyl compound is a competitive inhibitor (K-i = 0.76 mM). We have therefore synthesized various 4'- and 6'-O-alkyl de rivatives, some with reactive groups attached to an O-pentyl spacer, a nd tested these compounds as reversible and irreversible inhibitors of GlcNAc-TI. The 6'-O-(5-iodoacetamido-pentyl) compound is a specific t ime dependent inhibitor of the enzyme. Four other 6'-O-alkyl compounds showed competitive inhibition while the remaining compounds showed li ttle or no binding indicating that the electronic properties of the at tached O-pentyl groups influence binding.