The mechanism of the non-enzymatic breakdown of the tetrahedral intermediate of MurA (a carboxyvinyl transferase) was determined in order to better understand the corresponding enzyme catalyzed reaction. The breakdown was general acid-catalyzed, with simultaneous C-O bond cleavage and protonation at the bridging oxygen of the phosphate leaving group. The carboxyl was shown to play an important role in the breakdown, with its pKβ of 3.2 appearing in the rate π·π¦π³π΄πΆπ΄ pH profile. The product distribution varied with pH. At pH 1, the products were predominantly UDP-GIcNAc, pyruvate and phosphate. Increasing proportions of MurA ketal arising from intramolecular attack of the 4'-0H, and EP-UDP-GicNAc resulting from elimination were observed with higher pHs. The MurA ketal was structurally characterized, and shown to be analogous to the ketal formed by AroA, a related enzyme. pKβs corresponding to those expected of phosphate monoesters (1.6 and 6.2) were found in the product distribution π·π¦π³π΄πΆπ΄ pH profile. Observation of these pKβs supported the initial product of phosphate departure being an oxocarbenium ion-phosphate tight ion pair complex. A corresponding oxocarbenium ion intermediate in the enzymatic reaction could not be demonstrated. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22729 |
| Date | 09 1900 |
| Creators | Byczynski, Bartosz |
| Contributors | Berti, Paul, Chemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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