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601st MEETING, ABERDEEN 105 Epoxyalkyl peptide derivatives as active-site-directedinhibitors of asparagine N-glycosyltransferases ERNST BAUSE tnstitut f u r Biochemie, Universitat zu Koln, 0-5000 Koln, Federal Republic of Germany Synthetic peptides of defined structure represent excellent tools for the characterization of substrate specificities of asparagine N-glycosyltransferases (Bause, 1979; Bause & Hettkamp, 1979). By using this model approach, we recently demonstrated that triplet sequences of the type Asn-Xaa-Thr(Ser,Cys) are structural requirements for N-glycosylation, as was postulated some years ago by Marshall (1974). In addition, these studies revealed that the hydroxy-amino acid in this particular position of these tripeptides participates actively in the catalytic mechanism of transglycosylation. This occurs by promoting, in a kind of proton-relay system, the necessary hydrogen transfer from the p-amide of asparagine on to a corresponding basic group at the active site of the transferase (Bause & Legler, 1981). On the basis of this knowledge of the catalytic mechanism of asparagine N-glycosyltransferases, we have designed and synthesized two hexapeptides as potential active-site-directed inhibitors for this class of enzymes. Both compounds are derived from the basic sequence Arg-Asn-Gly-Yaa-Ala-Val-OMe, where Yaa represents epoxyethylglycine (I) and 2,3-epoxypropylglycine (11). Hence, these derivatives represent substrate analogues that are carrying a chemically reactive group at a strategically favoured position for a reaction with an amino acid side chain at the active site. They were prepared by epoxidation with p-chloroperbenzoic acid from their corresponding olefinic peptide precursors. The latter compounds were synthesized by Abbreviation used: OMe, methoxy (methyl ester). the solid-phase method as described by Erickson & Merrifield (1976). Preliminary experiments showed that neither compound I nor compound I1 was acting as a glycosyl acceptor under standard assay conditions, which usually generate soluble glycopeptides (Bause, 1979). However, when the particulate membrane fraction was pre-incubated with these derivatives in the presence of detergents, which are required to facilitate accessibility of the transferases, a time- and concentration-dependent loss of enzyme activity is observed with peptide I, whereas no inhibiting effect is produced by derivative 11. Partial protection against inhibition by peptide I could be obtained, when the preincubation experiments were carried out in the presence of the standard acceptor peptide Arg-Asn-Gly-Thr-Ala-Val-OMe. These observations suggest a specific reaction of inhibitor I at the active site of the transferases. The inhibitory effect exerted by compound I was less pronounced when the transferases were pre-incubated with the standard acceptor peptide before the addition of the inhibitor. As this treatment reduces the endogenous pool of glycosyl donor molecules, we conclude that the inactivation occurs only under conditions where glycosyl transfer is catalysed. The various experimental data are consistent with a mechanism of inactivation as outlined in Fig. l(a). For clarity, Fig. l ( b ) shows a model of the catalytic mechanism of N-glycosylation as proposed recently (Bause & Legler, 1981). We assume that, comparable with the hydroxy group of threonine or serine, the oxygen atom of the epoxy function is capable of interacting with the P-amide of asparagine via hydrogen bonding. As a result of this contact, the corresponding structure might be recognized as 'active conformation' and consequently be bound and glycosylated. The process of glycosylation is, after binding of the Dol-PP E-Enzyme J -------c2 I /CH, I h'~G1ycosyltransferase b Dol-PP-OS (b) 0 Dol-PP Tr \c/ !-Enzyme J OH '"rH ------__--- -- 0: I HCH I I H I 0 HC-CH, I I I \NAcY\cHAN/cTc/ I H d B A d B-Enzyme H\ o\c N-Glycosyltransferase b I /OS 2- H,. I HCH H I I I \N,cec/N,. I II H O 0 0: I I HC-CH, Abbreviations: Dol-PP, dolichyl diphosphate; Dol-PP-OS, dolichyl diphosphate oligosaccharide; B-Enzyme, base at the active site of N-glycosyltransferase. 11 I C\N/ CH \c/U CH L A O 1 Fig. 1. Proposed mechanism of inactivation of asparagine N-glycosyltransferases by the epoxyethylglycine-containinginhibitor peptide ( a ) and catalytic mechanism of N-glycosylation ( b )according to Bause & Legler ( I 981) VOl. H 106 epoxyalkyl peptide to the enzyme, initiated by the protonation of the oxiran ring giving rise to an intermediate with high alkylating potential. This structure is then stabilized, e.g. by a nucleophilic attack through the catalytically active base, which normally accepts the Bamide proton during catalysis. The final product of this reaction sequence is an inactivated enzyme containing the glycosylated inhibitor covalently attached via the epoxy function- This type of ~%ction mechanism implies that the N-glycosyltransferase catalyses its own inactivation in a kind of ‘suicide mechanism’. The various data support our present idea BIOCHEMICAL SOCIETY TRANSACTIONS of the functional role of the hydroxy amino acid during the process of N-glycosylation. Bause, E. (1979) FEBS Lett. 103,296299 Bause, E. He*amp, H. (1979) FEBs Lett. lm 341-344 Bause, E.Bt Legler, G.(1981)Biochem. J . 195, 639-644 Marshall, R. D.(1974) ~i~-~. so,.. symp. 40, 17-26 Erickson, B. W. & Medieid, R. B. (1976) in The Proteins (Neurath, H. & Hill, R.L.,eds.), vol. 2, pp. 255-562, Academic Press, London and New York 1983