Conformational and mechanistic studies of asparagine-linked glycosylation

Citation

Shannon, Karen Lynn (1992) Conformational and mechanistic studies of asparagine-linked glycosylation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/j6dq-0n18. https://resolver.caltech.edu/CaltechTHESIS:09082011-144447003

Abstract

In asparagine-linked glycosylation, a complex carbohydrate is transferred by oligosaccharyl transferase to an asparagine in the consensus triad -Asn-Xaa-Thr/Ser-. The mechanism of this transfer is not fully understood but appears to involve nucleophilic attack by the carboxyamido group of asparagine at the anomeric carbon of the sugar with concomittant displacement of dolichol pyrophosphate. Central to understanding the mechanism of N-glycosylation is ascertaining how the reactivity of a relatively poor nucleophile, the carboxyamido group, can be enhanced.

To increase the amide-nitrogen nucleophilicity, the amide resonance interaction must be disrupted. The necessity for a hydroxy amino acid two residues from the asparagine suggests that this residue may participate in a hydrogen bonding interaction. If the hydroxyl group is hydrogen bonded to the amide oxygen of the asparaginyl side chain, this interaction would increase the pKa of the amide protons through disruption of the amide resonance. Further, a general base could then abstract a proton, thereby increasing the nucleophility of the amide nitrogen.

If such a hydrogen bonding array is crucial to the mechanism of the transfer, the conformation of the peptidyl substrate must allow for the asparagine and threonine side chains to be in close proximity. If such a hydrogen bonding array is crucial to the mechanism of the transfer, the conformation of the peptidyl substrate must allow for the asparagine and threonine side chains to be in close proximity. If such a hydrogen bonding array is crucial to the mechanism of the transfer, the conformation of the peptidyl substrate must allow for the asparagine and threonine side chains to be in close proximity.

A series of tripeptides that satisfy the -Asn-Xaa-Thr/Ser-primary sequence requirement were synthesized and examined as potential acceptors in an oligosaccharyl transferase assay. It was demonstrated that the dominant solution conformation of substrates in glycosylation were Asx-tums. This conformation is stabilized by an interaction between the asparaginyl carboxyamido oxygen and the backbone amide proton of the hydroxy amino acid residue. The Asx-tum is a common structural motif in peptides and, in this case, brings the carboxyamido and hydroxyl groups within hydrogen bonding distance. Using constrained peptidyl analogs, the Asx-tum was demonstrated to be the bioactive conformation in N-glycosylation.

Further, peptidyl analogs were used to probe the mechanism of glycosylation. A mechanism which is consistent with both the conformational and kinetic data obtained in these studies is proposed.

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