Design, synthesis, and evaluation of unnatural amino acid-containing peptides as mechanistic probes and inhibitors of oligosaccharyltransferase.

Abstract

Oligosaccharyltransferase (OST, EC 2.4.1.119), is a membrane-bound, multimeric enzyme which catalyzes a key reaction in the formation of N-linked glycoproteins. During the OST-catalyzed reaction, the oligosaccharyl portion of Glc$\sb3$Man$\sb9$GlcNAc$\sb2$-PP-Dolichol is transferred to the $\beta$-amido group of an asparagine moiety in the growing polypeptide chain. The catalytic mechanism of OST is not clear, and unnatural amino acid-containing peptides have been synthesized and used to study this intriguing reaction. The carboxamide moiety that links the carbohydrate and protein moieties has been unambiguously determined to arise intact from asparagine by the use of chemically synthesized Bz- (4-$\sp{13}$C, $\sp{15}$N) Asn-Leu-Thr-NH$\sb2$ as an OST substrate. Bz- (4-$\sp{13}$C) Asn-Leu-Thr-NH$\sb2$ was also synthesized and used to evaluate a proposed mechanism of OST catalysis similar to glutamine-dependent amidotransferases using $\sp{15}$NH$\sb4$OAc as a potential external nucleophile. Analysis of NMR and MS spectra of the isotopically-labeled peptides and the resulting biosynthesized glycopeptides indicates that free $\sp{15}$NH$\sb3$ is not lost from the doubly labeled substrate during catalysis nor can exogenous $\rm\sp{15}NH\sb3$ intercept any of several postulated enzyme-bound species. These results indicate that OST-catalyzed glycosylation does not follow a mechanism involving the transient generation of exchangeable NH$\sb3$. 5-diazo-4-oxo-L-norvaline, 4-oxo-L-norvaline, and S-methanesulfinyl-L-alanine have been incorporated into three separate tripeptides wherein these non-natural amino acids replace Asn in a known tripeptide substrate of OST. The three synthetic tripeptides showed no activity as substrates and only weak activity as inhibitors of OST at concentrations that were 10-35 times the K$\rm\sb{m}$ for the corresponding Asn-containing tripeptide substrate. A benzophenone photophore was incorporated into peptides as potential photoaffinity labels of OST. Bz-Asn-Bpa-Thr-NH$\sb2$ and Bz-Asn-Lys ((4-Bz)Bz) -Thr-NH$\sb2$ were found to be good alternate substrates. They were also good competitive inhibitors vs standard peptide substrate ($\sp{14}$C) Bz-Asn-Leu-Thr-NH$\sb2$ and their K$\rm\sb{i}$ values were determined.