Rates of Chemical Reactions Embedded in a Metabolic Network by Dissolution Dynamic Nuclear Polarisation NMR

International audienceThe isomerisation of 6-phosphogluconolactones and their hydrolyses into 6-phosphogluconic acid form a non enzymatic side cycle of the pentose-phosphate pathway (PPP) in cells. We show that dissolution dynamic nuclear polarization can be used for determining the kinetic rates of the involved transformations in real time. It is found that the hydrolysis of both lactones is significantly slower than the isomeration process, thereby shedding new light onto this subtle chemical process

Application of Circular Dichroism Spectroscopy to the Analysis of the Interaction Between the Estrogen Receptor Alpha and Coactivators: The Case of Calmodulin

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Backbone H(N), N, C(alpha), C', and C(beta) assignment of the 6-phosphogluconolactonase, a 266-residue enzyme of the pentose-phosphate pathway from human parasite Trypanosoma brucei

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β,γ-Diamino acid: an original building block for hybrid α/γ-peptide synthesis with extra hydrogen bond donating group.

International audience: Using a β,γ-diamino acid, several small hybrid α/γ peptides have been synthesized and their conformations investigated through extensive NMR studies and molecular dynamics. A tripeptide and a tetrapeptide have thus shown several hydrogen bonds in solution, including a 13-membered ring involving the β-nitrogen

Methodological Study of the Peptide Coupling of the N-desactivated Stable Proline Surrogate CF3-pseudoproline

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Incorporation of CF3-Pseudoprolines into Peptides: a Methodological Study.

International audience: The peptide coupling reactions allowing the incorporation of trifluoromethyl substituted oxazolidine-type pseudoprolines (CF3-psiPro) into peptide chains have been studied. While standard protocols can be used for the peptide coupling reaction at the C-terminal position of the CF3-psiPro, acid chloride activation has to be used for the peptide coupling reaction at the N-terminal position to overcome the decrease of nucleophilicity of the CF3-psiPro. We demonstrate that the N-amidification of a diastereomeric mixture of CF3-psiPro using Fmoc protected amino acid chloride without base gave the corresponding dipeptides as a single diastereomer (6 examples). The ratio of the cis and trans amide bond conformers was determined by NMR study, highlighting the role of the Xaa side chains in the control of the peptide backbone conformation. Finally a tripeptide bearing a central CF3-psiPro has been successfully synthesized

Toward Quantitative Measurements of Enzyme Kinetics by Dissolution Dynamic Nuclear Polarization

Dissolution dynamic nuclear polarization (D-DNP) experiments enabled us to study the kinetics of the enzymatic phosphorylation reaction of glucose to form glucose-6-phosphate (G6P) by hexokinase (HK), with or without the presence of an excess of G6P, which is known to be an inhibitor of the enzyme. Against all expectations, our observations demonstrate that the phosphorylation of both alpha and beta glucose anomers occurs with comparable kinetics. The catalytic constant of the reaction was estimated based on a simple kinetic model tailored for hyperpolarized systems

New insights into the structural and dynamics properties of collagen model peptides using CD spectroscopy, MD simulations and innovative NMR approaches

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Insights Into the Enzymatic Mechanism of 6-Phosphogluconolactonase from Trypanosoma brucei Using Structural Data and Molecular Dynamics Simulation

Trypanosoma brucei is the causative agent of African sleeping sickness. Current work for the development of new drugs against this pathology includes evaluation of enzymes of the pentose phosphate pathway (PPP), which first requires a clear understanding of their function and mechanism of action. In this context, we focused on T brucei 6-phosphogluconolactonase (Tb6PGL), which converts delta-6-phosphogluconolactone into 6-phosphogluconic acid in the second step of the PPP. We have determined the crystal structure of Tb6PGL in complex with two ligands, 6-phosphogluconic acid and citrate, at 2.2 angstrom and 2.0 angstrom resolution, respectively. We have performed molecular dynamics (MD) Simulations on Tb6PGL in its empty form and in complex with delta-6-phosphogluconolactone, its natural ligand. Analysis of the structural data and MID simulations allowed LIS to propose a detailed enzymatic mechanism for 6PGL enzymes. (C) 2009 Elsevier Ltd. All rights reserved