Use this URL to cite or link to this record in EThOS:
Title: Mechanistic studies of 4-amino-4-deoxychorismate synthase
Author: Bulloch, Esther Marcella MacLeod
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
Availability of Full Text:
Full text unavailable from EThOS.
Please contact the current institution’s library for further details.
This thesis describes mechanistic and structural studies of Escherichia coli 4-amino-4-deoxychorismate synthase (ADCS). ADCS catalyses the conversion of chorismate to 4-amino-4-deoxychorismate, which is the first committed step in folate biosynthesis. The enzyme consists of a glutamine amidotransferase, PabA, and a chorismate aminating subunit, PabB. ADCS belongs to a family of closely related chorismate-utilising enzymes that also includes anthranilate synthase. In complement to the studies of ADCS, some research was also conducted into the mechanism of the reaction catalysed by anthranilate synthase. Site directed mutagenesis studies were carried out on active site residues of ADCS and anthranilate synthase with the aim of probing the mechanism of each enzyme. Kinetic parameters were determined for active mutants, and the products of the reactions catalysed by each mutant were examined by NMR spectroscopy. Lys-274 or PabB was identified as having an important catalytic role in the reaction catalysed by ADCS. The studies of anthranilate synthase indicate that an active site glutamate residue, which is also conserved in ADCS, assists in the elimination of the C4 hydroxyl from chorismate during catalysis. ADCS is thought to be the final in vivo target of the antibiotic (6S)-60fluoroshikimate. (6S)-6-Fluoroshikimate is converted to 2-flurochorismate by enzymes of the shikimate pathway. In this thesis it is shown that 2-fluorochrosimate inactivates ADCS by causing an irreversible covalent modification of Lys-274 of PabB. This result supports by hypothesis that Lys-274 is the active site nucleophile of the reaction catalysed by PabB. The formation of a covalent intermediate on PabB during the ADCS reaction was directly directed for the first time using electrospray ionisation mass spectrometry. Control experiments indicated that the covalent intermediate observed is competent in the ADCS reaction, and that the intermediate most likely forms on Lys-274. The glutamyl-thioester covalent intermediate of the reaction catalysed by PabA was also detected.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available