Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.776074
Title: Thioredoxin reductase and dihydrolipoamide dehydrogenases of Plasmodium falciparum
Author: McMillan, Paul James
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2006
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Abstract:
Plasmodium falciparum is an obligate intracellular protozoan parasite and is the causative agent of malaria, which infects 270 million people and causes 2-3 million human deaths each year. During the erythrocytic stages of their life cycle the parasites are exposed to reactive oxygen species generated by the host and their own metabolism. As a result the parasites have developed efficient antioxidant defence mechanisms, which include the thioredoxin system. A key component of the thioredoxin system is the flavin adenine dinucleotide (FAD)-dependent disulphide oxidoreductase thioredoxin reductase (PfTrxR), which provides the reducing power for the system using NADPH. PfTrxR is a high Mr TrxR and has been genetically and chemically validated as a potential drug target by targeted gene dismption and the identification of several specific inhibitors. PfTrxR contains three redox active centres (FAD, Cys88/Cys93 and Cys535/Cys540) that are in redox communication. This study examined the catalytic mechanism (i.e. the transfer of reducing equivalents between these redox active centres) of PfTrxR by employing pre-steady state kinetics of the reductive (reduction of PfTrxR by NADPH) and oxidative half reaction (oxidation of reduced PfTrxR by thioredoxin). This identified that the PfTrxR protein conformed to the catalytic mechanism for high Mr TrxR, which was proposed from studies on the Drosophila melanogaster TrxR (DmTrxR). However, a few key differences were observed between the proteins from the two organisms. Firstly, the reductive half reaction of PfTrxR was observed to proceed at a faster rate than that of DmTrxR. Secondly, the oxidative half reaction was observed to proceed at a slower rate than that of DmTrxR protein. Finally, PfTrxR was shown not to cycle between a two (EH2) and four (EH4) electron reduced species in vitro (as demonstrated in DmTrxR) and was observed to be almost fully oxidised following reaction with excess thioredoxin. However, the rate of oxidation from the two electron reduced (EH2) to oxidised (Eox) forms was extremely slow and would probably not be relevant in vivo due to the high NADPH concentration. The role of conserved PfTrxR active site residues was investigated by producing site directed mutants and analysing them by steady state kinetics, anaerobic static titrations and pre-steady state kinetics. Particular attention was paid to the residues (His509 and Glu514 in PfTrxR) contained in the acid/base catalyst motif (HPXXXE) that has been identified and investigated in other disulphide oxidoreductase proteins. This demonstrated that both of these residues played crucial, yet distinct, roles in the transfer of reducing equivalents between the redox active centres in PfTrxR.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.776074  DOI:
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