STRUCTURAL AND FUNCTIONAL-ANALYSIS OF PEPTIDYL OLIGOSACCHARYL TRANSFERASE INHIBITORS

The peptide -Cys((2)))-Thr((3))-Val((4))-Thr((5))-Nph((6))-NH2 was pre viously shown to be a slow, tight-binding inhibitor (K-i = 37 nM) of t he yeast oligosaccharyl transferase (OT) [Hendrickson et al. (1996) J. Am. Chem. Soc. 118, 7636-7637]. This enzyme catalyzes the transfer of a carbohydrate moiety to an asparagine residue in the consensus seque nce Asn-Xaa-Thr/Ser. Herein we present a study of the contribution of the residues in positions 1, 3, 4, and 5 to OT binding. Replacement of the threonine (residue 3) by valine or (S)-2-aminobutyric acid dramat ically reduced the potency of the inhibitor while, surprisingly, the i ncorporation of an additional methylene into the side chain of residue 1 [(S)-2,3-diaminobutyric acid changed to ornithine] had very little effect. Variants with acidic, basic, hydrophilic/polar, and hydrophobi c side chains in positions 4 and 5 were also evaluated for both yeast and porcine liver OT inhibition. This aspect of the study reveals that basic (lysine) and acidic (glutamic acid) residues are detrimental to the binding, whereas hydrophobic (valine) and polar/hydrophilic (thre onine) residues are both well tolerated. The kinetic behavior of subst rate analogs (1))-Cys((2)))-Thr((3))-Xaa((4))-Yaa((5))-Nph-NH2] corres ponding to inhibitors of weak, medium, and strong potency was also exa mined in order to provide insight into the nature of these inhibitors.