Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.529672
Title: Kinetics and specificity of nicotinamide nucleotide binding to the dIII component of transhydrogenase from Rhodospirillum Rubrum
Author: Huxley, Lucinda
Awarding Body: University of Birmingham
Current Institution: University of Birmingham
Date of Award: 2011
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Abstract:
Transhydrogenase is an enzyme located in the cytoplasmic membrane of bacteria or the inner membrane of animal mitochondria. Using the energy of the proton electrochemical gradient (Δp), transhydrogenase translocates protons across the membrane whilst undergoing its redox reaction, in which hydride ion equivalents are transferred from NADH to NADP+ producing NAD+ and NADPH. Transhydrogenase comprises three components; dI binds NA(H), dIII binds NADP(H) and dII spans the membrane. Transhydrogenase is thought to function by way of a binding-change mechanism, which involves “open” and “occluded” conformations of the enzyme. In the open conformation, nucleotides can readily bind and dissociate from the enzyme but the hydride transfer reaction is blocked. In the occluded conformation, hydride transfer is permitted but the binding and release of nucleotides is blocked. Hydride transfer and proton translocation are coupled. The coupling is not well understood due to the lack of structural information about the membrane-spanning dII component. However, it is believed to involve conformational changes of the enzyme, particularly the dII and dIII components, resulting in the switch between the open and occluded conformations. Enzyme assays and tryptophan fluorescence experiments using apo-dIII in complex with dI revealed two features: Firstly, the binding of NADP(H) to dIII is very slow and is probably limited by the conversion from the occluded to the open conformation. Since the switch between the occluded and open conformations is thought to be central in the coupling of hydride transfer and proton translocation, the results presented here give an insight into the binding-change mechanism of transhydrogenase. Secondly, NAD(H) is able to slowly bind into the NADP(H)-binding site of dIII (the “wrong” site). This brought into question the specificity of the dIII component of transhydrogenase for NADP(H). The significance and likelihood of NAD(H) binding to dIII in the intact enzyme in the living cell are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.529672  DOI: Not available
Keywords: QH301 Biology ; QR Microbiology
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