Insights into the Mechanism of Asparagine-Linked Glycosylation: Kinetic Studies with Substrates and Inhibitors
<p>Investigations into the mechanism of action of oligosaccharyl transferase (OT), the enzyme that catalyzes the first committed step in the biosynthesis of all asparagine-linked glycoproteins, were conducted. Research focused on kinetic studies with small reactive molecules and tripeptide sub...
| Summary: | <p>Investigations into the mechanism of action of oligosaccharyl transferase (OT), the enzyme that catalyzes the first committed step in the biosynthesis of all asparagine-linked glycoproteins, were conducted. Research focused on kinetic studies with small reactive molecules and tripeptide substrates and inhibitors; these experiments were designed to specifically probe the OT active site. Commercially available chemical modification agents were used to identify a reactive cysteine residue in or near the oligosaccharide binding site. Based on this observation, a novel biotinylated sufhydryl modification reagent, methyl <i>N</i>-biotinoyl(aminoethane)thiolsulfonate (BMTS), was developed. BMTS selectively modified the Wbp1p subunit of OT, thereby identifying this subunit as the site of the reactive cysteine and all or part of the oligosaccharide binding site.</p>
<p>The role of the required divalent metal cofactor was examined through a series of parallel kinetic experiments on two substrates for OT: the asparagine-containing tripeptide Bz-Asn-Leu-Thr-NHMe and the corresponding thioasparagine tripeptide Bz-Asn(γS)-Leu-Thr-NHMe. These substrates were evaluated with OT which had been treated to remove metal and independently reconstituted with four different metal cations. The sizes and thiophilicities of the metal cations had a different, but direct impact on the kinetic behavior of each of the two peptides. These results place the metal cofactor in proximity to the peptide substrate during catalysis and suggest that it may play a direct mechanistic role. In addition, a detailed analysis of the glycopeptide product of the thioasparagine tripeptide suggests that OT plays a specific role in directing the regiochemistry of asparagine glycosylation.</p>
<p>Through an iterative process, a new class of competitive inhibitors for OT was designed which exhibit nanomolar binding constants and are the first reported potent inhibitors for OT. The slow, tight binding inhibition observed with these peptides suggests that they may mimic the transition state of the glycosylation reaction. Nine irreversible peptidyl inactivators for OT were also evaluated; these affinity labels were designed to probe the peptide binding site of the enzyme. Based on the results with these inactivators, a proposal for the design of more potent affinity labels for OT is presented.</p> |
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