25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

Fluxomic studies by in cell and in vitro Dissolution-Dynamic Nuclear Polarization NMR

29 Aug 2019, 12:30
30m
Max Kade Auditorium (Henry Ford Building)

Max Kade Auditorium

Henry Ford Building

Talk In-vivo and in-cell In-vivo

Speaker

Mr David Guarin (Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France)

Description

Since the invention of dissolution Dynamic Nuclear Polarization (DDNP)$^{1}$ and the dramatic signal enhancement it provides in solution-state NMR, a wide range of applications have emerged. In particular, this improvement opens new avenues for the study of fast dynamical processes such as chemical reactions, with a time resolution smaller than a second. DDNP has proven of particular interest in the study of such metabolic processes as enzymatic reactions that are affected by many factors, such as dietary or environmental. These reactions can now be studied in vitro and in cell, providing access to the nature and degree of possible cell impairment.

We have initiated kinetic studies of enzymatic reactions using DDNP on the oxidative stage of the Pentose Phosphate Pathway (oxPPP)$^2$, one of the crucial metabolic pathways in cells. OxPPP produces NADPH, one of the main sources of reductive power in the cell. It comprises three enzymes: Glucose-6-Phosphate dehydrogenase (G6PDH), 6-Phosphogluconolactonase and 6-Phosphogluconic acid dehydrogenase.

By studying the enzymatic cascade involving Hexokinase and G6PDH, we could extract the relevant kinetic parameters involved in the process. The complexity of the kinetic pathway and the large number of model parameters makes it a particularly demanding task that requires the repeatability of the experiments. Fitting the signal build-up curves of the multiple metabolites allowed us to extract the relevant kinetic parameters. This showed satisfactory correlation with the enzyme activities used in the experiments.

In parallel, in cell experiments performed on E. coli allowed to provide a global ans quantitative view of Glucose metabolism in function of the cell-growth and cell stress conditions. In particular, the differential $\alpha / \beta$ anomeric Glucose uptake by the cells appeared as an indicator of the relative activities of Glycolysis and PPP.

  1. Ardenkjær-Larsen et al. PNAS, (2003), 10158–10163.

  2. A. Sadet et al. Chem.Eur.J. (2018), 4, 5456–5461.

Primary authors

Mr David Guarin (Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France) Ms Jessie Mosso (Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France) Mr Mathieu Baudin (Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France) Mr Dennis Kurzbach (University Vienna, Faculty of Chemistry, Institut of biological chemistry, Whringer Straße 38, 1090 Vienna, Austria - Vienna ) Mr Emeric Miclet (Sorbonne Université, École normale supérieure, PSL University, CNRS, Laboratoire des biomolécules, LBM, 75005 Paris, France) Dr Daniel Abergel (Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France)

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