Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484967
Title: Investigations into lipoic acid biosynthesis
Author: Bryant, Penny
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2007
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Abstract:
The protein lipoyl synthase (LipA) is essential for lipoic acid biosynthesis via sulfur insertions at the C6 and C8 positions of a protein-bound oetanoyl group. LipA from Escherichia coli binds two .[4Fe-4S]1+12+ and is a member of the radical SAL\1 superfamily of proteins. To facilitate mechanistic investigations into LipA dependent lipoyl synthesis, a novel in vitro assay has been developed which makes use of synthetic peptide substrates. These peptides contain an N(~)-octanoyl lysine residue, corresponding in sequence to the lipoyl binding domain of the E2 subunit of pyruvate dehydrogenase. The activity LipA from Sulfolobus solfataricus was measured using these substrate analogues. The optimal temperature for the S. solfataricus LipA dependent formation of the lipoyl group was found to be 60°C. Time dependent activity of LipA has been investigated over a wide range of temperatures (23-60 DC) and rate constants approximated for the observed rates of loss of octanoyl starting material and lipoyl formation at each temperature. This has allowed calculation of rate constants for the overall rate. kinetic analysis over a range of temperatures has allowed the activation energy for overall lipoyl formation (47.2 ± 5.4 kJ/mol) and for sulfur insertion at C6 (38.3 ± 4.9 kJ/mol) and C8 (46.9 ± 5.7 kJ/mal) to be determined In all experiments using ofS. solfataricus LipA, reconstitution ,vith exogenous iron and sulfide was found to be essential for activity and spectroscopic studies have been carried out which show that S. solfataricus LipA can bind two [4Fe-4S] 1+/2+ and therefore closely resembles E. coli LipA. The mechanistic roles of these clusters have been investigated_and EPR spectroscopy suggested that SALVI bound to at least one of
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.484967  DOI: Not available
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