Mechanism of Allosteric Behaviour and Substrate Inhibition of Lactococcus Lactis Prolidase
| dc.contributor.advisor | Tanaka, Takuji | |
| dc.contributor.committeeMember | Grochulski, Pawel | |
| dc.contributor.committeeMember | Korber, Darren | |
| dc.contributor.committeeMember | Tyler, Robert | |
| dc.contributor.committeeMember | Sanders, David | |
| dc.creator | Xu, Shangyi | |
| dc.date.accessioned | 2021-09-27T16:46:29Z | |
| dc.date.available | 2023-09-27T06:05:08Z | |
| dc.date.created | 2021-09 | |
| dc.date.issued | 2021-09-27 | |
| dc.date.submitted | September 2021 | |
| dc.date.updated | 2021-09-27T16:46:29Z | |
| dc.description.abstract | Prolidase (E.C 3.4.13.9) is an enzyme that specifically hydrolyzes Xaa-Pro dipeptides into free amino acids. Allosteric behaviour and substrate inhibition are unique features of wild-type (WT) Lactococcus lactis prolidase (Llprol). Our previous mutagenesis studies and kinetic studies indicate such features are related to inter-subunit interactions between loop 36-40 and residues near the active site of Llprol. D36S and R293S Llprol mutants show no allosteric behaviour, and substrate inhibition was not detected in Llprol H38S and R293S. This thesis study investigated the structures of D36S, H38S, and R293S Llprol mutants with X-ray crystallography. The crystal structures of the mutants were solved, and they were compared with the structure of WT Llprol. It was found that the two monomers that formed the Llprol homodimer are at a closer distance in all the allosteric variants of Llprol (WT and H38S). Interatomic distances between the alpha carbons on loop 36-40 from one of the monomers and His 204 at the corresponding active center from the other monomer were measured. In WT Llprol and Llprol H38S, the average distances between His 204 and loop 36-40 were 15.64 and 15.19 Å, respectively. However, in non-allosteric Llprol D36S and R293S, this distance increased to 16.01 and 17.13 Å, respectively. Also, the percentage interface area of the two monomers was larger in WT Llprol and Llprol H38S dimers (5.77% and 5.07%, respectively) than the interface area of non-allosteric mutants (D36S and R293S; 4.67% and 4.95%, respectively). We suspect the entryway of the substrate is blocked when Llprol adapts to the conformation featuring two closer monomers, so allosteric behaviour was observed. This study also identified a possible pseudo-binding site (His 38, Arg 40, Arg 293, and His 303) near the active center of Llprol. This pseudo site had a similar amino acid composition compared to the active site, consisting of two histidine and one arginine. The effectiveness of this proposed pseudo site was supported with substrate docking modelling. It is proposed that substrate inhibition of WT Llprol and Llprol D36S is due to the substrate-binding at the pseudo site when substrate concentration is higher than 4 mM. Substrate inhibition is eliminated in Llprol H38S and R293S since they have impaired pseudo sites. In conclusion, it is proposed that Llprol exhibits allosteric behaviour because it exists in multiple conformations with different substrate affinity, and the pseudo binding at high substrate concentration leads to the substrate inhibition of Llprol. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | https://hdl.handle.net/10388/13614 | |
| dc.subject | Prolidase | |
| dc.subject | Proline-containing peptides | |
| dc.subject | Allosteric behaviour | |
| dc.subject | Substrate inhibition | |
| dc.subject | Inter-subunit interaction | |
| dc.subject | Structure-function relationship | |
| dc.title | Mechanism of Allosteric Behaviour and Substrate Inhibition of Lactococcus Lactis Prolidase | |
| dc.type | Thesis | |
| dc.type.material | text | |
| local.embargo.terms | 2023-09-27 | |
| thesis.degree.department | Food and Bioproduct Sciences | |
| thesis.degree.discipline | Food Science | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.Sc.) |