Evaluation of wild type and mutants of β-Glucuronidase (GUS) against natural and synthetic substrates
| dc.contributor.advisor | Sanders, David | en_US |
| dc.contributor.committeeMember | Palmer, David | en_US |
| dc.contributor.committeeMember | Urquhart, Stephen | en_US |
| dc.contributor.committeeMember | Mueller, Jens | en_US |
| dc.contributor.committeeMember | Chelico, Linda | en_US |
| dc.creator | Tiwari, Siddharth | en_US |
| dc.date.accessioned | 2014-06-13T12:00:15Z | |
| dc.date.available | 2014-06-13T12:00:15Z | |
| dc.date.created | 2014-04 | en_US |
| dc.date.issued | 2014-06-12 | en_US |
| dc.date.submitted | April 2014 | en_US |
| dc.description.abstract | Modifying substrate specificity of β-glucuronidase (GUS) would be helpful in various enzyme prodrug systems in delivering drug dose to the site of action in the cancer treatment. Due to the presence of endogenous enzyme in human tissues, GUS-based Antibody-Directed Enzyme Prodrug Therapy (ADEPT) requires a novel substrate to avoid undesirable systemic activation. GUS is a glycosyl hydrolase, highly specific towards the glucuronide derivatives. It catalyzes the glycosidic cleavage of β-D-glucuronides to β-D-glucuronic acid and aglycone moiety. In order to gain insight on the substrate specificity of GUS, C6 carboxyl group of glucuronic acid was modified to C6 carboxamide (amide derivative). We have examined amide derivatized substrates with a variety of different aglycone groups including p-nitrophenyl, phenyl and 4-methylumbelliferone to further probe the activity profile of GUS. In an effort to optimize GUS activity, docking studies have been performed which indicated that amino acid point mutations near C6 carboxyl group of glucuronic acid could improve binding of the derivatized substrates. As a result point mutations to Arg-562 and Lys-568 which make the active site less positively charged either by glutamine or glutamate lead to an enzyme with much lower native substrate activity but abolished activity for the amide-derivatized substrate. This research study showed that there is still a further need of finding appropriate mutations required to make glucuronamide a better substrate for the mutated version of GUS. | en_US |
| dc.identifier.uri | http://hdl.handle.net/10388/ETD-2014-04-1541 | en_US |
| dc.language.iso | eng | en_US |
| dc.subject | β-glucuronidase (GUS), Escherichia coli (E. coli), Glucaro-δ-lactam (GDL), p-nitrophenyl-β-D-Glucuronide (PNPG), p-nitrophenyl-β-D-Glucuronamide (PNPGun) | en_US |
| dc.title | Evaluation of wild type and mutants of β-Glucuronidase (GUS) against natural and synthetic substrates | en_US |
| dc.type.genre | Thesis | en_US |
| dc.type.material | text | en_US |
| thesis.degree.department | Chemistry | en_US |
| thesis.degree.discipline | Chemistry | en_US |
| thesis.degree.grantor | University of Saskatchewan | en_US |
| thesis.degree.level | Masters | en_US |
| thesis.degree.name | Master of Science (M.Sc.) | en_US |