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Etudes de cinétiques enzymatiques par polarisation dynamique nucléaire avec dissolution (D-DNP) : application à l'étape oxydative de la voie des pentoses phosphates (PPP)

Abstract : The Pentose Phosphate Pathway (PPP) is one of the main pathways of cellular metabolism. This metabolic pathway is composed of two enzymatic cascades: one is an oxidative pathway, and the other is non-oxidative. The oxidative branch of PPP produces a cofactor, NADPH, which is responsible for the detoxification process of the cell due to its reducing activity, and is also a precursor of various biosynthesis such as lipogenesis. A dysfunction of one of the three enzymes that make up this step of PPP can lead to cell death. Thanks to a new method, Dissolution Dynamic Nuclear Polarization (D-DNP), which features a sensitivity gain by a factor 10,000 compared to standard liquid-state NMR, the quantification of kinetic parameters under physiological conditions, in cell, becomes possible.In this thesis, we add to the scientific library a new model of quantification of kinetic parameters, and the possibility of studying an enzymatic cascade composed of 3 enzymes by D-DNP measurements. Based on these experiments, the selectivity of the first enzyme in the oxidative pathway, G6PD, for one of the two glucose-6-phosphate anomers, was confirmed. The antioxidant role of the second PPP enzyme, 6PGL, was equally studied. To carry out these studies, a method of synthesis and purification of the different substrates of each enzyme has been developed. The very first inhibitor of 6PGL has also been tested. Preliminary experiments on Trypanosoma brucei, a parasite responsible for sleeping sickness, indicate that glucose penetration inside cells is the limiting kinetic step for its conversion.
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https://tel.archives-ouvertes.fr/tel-01893014
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Submitted on : Thursday, October 11, 2018 - 1:01:45 AM
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  • HAL Id : tel-01893014, version 1

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Aude Sadet. Etudes de cinétiques enzymatiques par polarisation dynamique nucléaire avec dissolution (D-DNP) : application à l'étape oxydative de la voie des pentoses phosphates (PPP). Chimie analytique. Université Pierre et Marie Curie - Paris VI, 2017. Français. ⟨NNT : 2017PA066217⟩. ⟨tel-01893014⟩

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