EPOXYETHYLGLYCYL PEPTIDES AS INHIBITORS OF OLIGOSACCHARYLTRANSFERASE - DOUBLE-LABELING OF THE ACTIVE-SITE

Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethyl glycine in the Asn-Xaa-Thr glycosylation tripler. Incubation of the en zyme in the presence of Dol-PP-linked [C-14]oligosaccharides and the N -3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both o f which are involved in the catalytic activity. Labelling of both subu nits was blocked competitively by the acceptor peptide N-benzoyl-Asn-G ly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-alpha,gamma-diaminobut yric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy -inhibitor by replacing asparagine with glutamine. Our data clearly sh ow that double-labelling is an active-site-directed modification, invo lving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Doubl e-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the co urse of which OST catalyses its own 'suicide' inactivation, is more li kely, as suggested by indirect experimental evidence. The syn-catalyti c mechanism corresponds with our current view of the functional role o f the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation [Bause, Breuer and Peters (1995) Bioc hem. J. 312, 979-985].