Please use this identifier to cite or link to this item:
http://hdl.handle.net/11375/18935
Full metadata record
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | Berti, Paul | - |
dc.contributor.author | Azhikannickal, Vincent | - |
dc.date.accessioned | 2016-03-14T19:38:40Z | - |
dc.date.available | 2016-03-14T19:38:40Z | - |
dc.date.issued | 2016 | - |
dc.identifier.uri | http://hdl.handle.net/11375/18935 | - |
dc.description.abstract | Escherichia coli AlkA (3-methyladenine DNA glycosylase II) is a DNA repair enzyme that can excise both damaged and undamaged nucleobases. To determine whether AlkA’s excision of neutral purines is facilitated by general- acid catalysis (i.e., protonation of the nucleobase), pH vs. kcat and pH vs. kcat/KM profiles for hypoxanthine excision were generated. Each profile revealed acid catalysis and a single pKa of 5.7 ± 0.1 and 5.1 ± 0.2, respectively. Mutants of ionizable and hydrogen-bonding active site residues – Y273F, W272F, Y222F, R244M, and R22M – demonstrated at most a 4-fold reduction in hypoxanthine excision, so these residues were not involved in purine protonation. A dependence on buffer concentration was observed in the mutants at pH 7 and in wild-type AlkA at pH 7 and 8 but not at pH < 7. Solvent deuterium kinetic isotope effects (KIEs) at pH 6 and 7 showed that proton transfer was rate-limiting at pH 7 when [DNA] > KM (KM = 1.0 μM). AlkA could not bind adenine, hypoxanthine, and nicotinamide along with a transition state-mimicking pyrrolidine DNA. Therefore, AlkA does not interact specifically with the nucleobase to protonate it. To determine the structure of AlkA’s transition state, methods for primary and secondary KIE measurement were developed, but none of the methods produced precise KIE values (95% confidence interval < 0.005). To determine the structure of the transition state of the cognate non-enzymatic reaction, the acid-catalyzed hydrolysis of 2’-deoxyinosine 5’-monophosphate, the 1’-3H, 4’-3H, 5’-3H2, 1’-14C, and 7-15N KIEs were measured and were 1.25 ± 0.02, 0.94 ± 0.01, 0.99 ± 0.02, 0.997 ± 0.004, and 0.99 ± 0.05, respectively. Despite being consistent with an oxacarbenium ion-like transition state, these values would not be precise enough to distinguish between potential transition state models. | en_US |
dc.language.iso | en | en_US |
dc.title | Towards the Mechanisms of AlkA-Catalyzed and Acid-Catalyzed 2’-Deoxyinosine Hydrolysis: Investigations Into Leaving-Group Activation and Transition State Structure | en_US |
dc.type | Thesis | en_US |
dc.contributor.department | Chemistry and Chemical Biology | en_US |
dc.description.degreetype | Thesis | en_US |
dc.description.degree | Doctor of Philosophy (PhD) | en_US |
dc.description.layabstract | When DNA, the genetic material in all living things, is chemically damaged, the health of the cell and its daughter cells is affected. To protect against DNA damage, proteins are produced by all cells to repair DNA. These proteins usually perform chemical reactions on a single type of damage in DNA, but Escherichia coli AlkA (3-methyladenine DNA glycosylase II) reacts with several types of damage. To study how AlkA participates in these varied reactions, its behaviour was examined under different conditions. The results show that it does not closely interact with the nucleobase of the damaged DNA. Additional aspects of the reaction in the presence and absence of AlkA were studied using an experimental method in which AlkA reacts with radioactive DNA. Attempts to use this method did not yield data that were reproducible enough to reveal an even more detailed picture of this reaction. | en_US |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
Azhikannickal_Vincent_finalsubmission201602_PhD.pdf | Thesis | 4.47 MB | Adobe PDF | View/Open |
Items in MacSphere are protected by copyright, with all rights reserved, unless otherwise indicated.