| This research has focused on determining if the enzyme oligosaccharyl-transferase (OST, EC 2.4.1.119) operates through an electrophilic-activation mechanism. OST catalyzes the transfer of a saccharide unit from a lipid-phosphate donor onto the beta-amido group of an asparagine moiety of a growing polypeptide chain, forming an N-linked glycopeptide. To help understand the means by which OST catalyzes this reaction, substrate analogues were designed as mechanistic probes. A novel peptide acceptor analogue, Asparagine-Lysine(Biotin)-Threonine was designed to allow development of a faster and more facile biotin-capture method of monitoring the OST reaction. Donor substrate analogues were also designed to contain either a proton or deuterium at the reaction site, enabling detection of the key oxocarbenium ion which defines an electrophilic activation mechanism. These donor analogues incorporated a distinct unstable isotopic label on the protio- and deuterio- substrates that could report on the reaction progress and be used in conjunction with the biotin-capture assay. The synthesis of the radio-labeled donor along with the biotinylated-tripeptide allowed for development of a biotin-capture assay for OST, as well as the determination of alpha-secondary kinetic isotope effects which could provide evidence to elucidate the mechanism of OST catalysis. Unfortunately, the OST system was to complex to allow for detection of a kinetic isotope effect on the reaction, indicating that much more work is needed to elucidate the mechanism of this enzyme. |
