Structural and functional analysis of peptidyl oligosaccharyl transferase inhibitors

被引:13
作者
Kellenberger, C [1 ]
Hendrickson, TL [1 ]
Imperiali, B [1 ]
机构
[1] CALTECH,DIV CHEM & CHEM ENGN,PASADENA,CA 91125
关键词
D O I
10.1021/bi971465d
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The peptide cyclo(hex-Amb((1))-Cys((2)))-Thr((3))-Val((4))-Thr((5))-Nph((6))-NH2 was previously shown to be a slow, tight-binding inhibitor (K-i = 37 nM) of the 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 sequence 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 dramatically reduced the potency of the inhibitor while, surprisingly, the incorporation 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 hydrophobic 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 (threonine) residues are both well tolerated. The kinetic behavior of substrate analogs [cyclo(hex-Asn((1))-Cys((2)))-Thr((3))-Xaa((4))-Yaa((5))-Nph-NH2] corresponding to inhibitors of weak, medium, and strong potency was also examined in order to provide insight into the nature of these inhibitors.
引用
收藏
页码:12554 / 12559
页数:6
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