Caractérisation des variations structurales de la créatine kinase à l'aide de sondes spectroscopiques et de l'échange H/D couplé à la spectrométrie de masse

Ce travail concerne un enzyme homodimérique, la créatine kinase cytosolique de muscle de lapin (CK-MM). L'utilisation combinée de méthodes biophysiques et de la technique d'échange H/D couplé à la protéolyse et à la spectrométrie de masse renseigne sur le processus de dépliement-repliement de la CK-MM dénaturée par le chlorure de guanidinium et sur sa dynamique structurale en présence ou non de substrats. La protéinase K clive la CK-MM en un segment N-terminal K1 et un segment C-terminal K2. L'analyse du repliement de cette protéine (K1K2)2 a permis de montrer que le domaine C-terminal joue un rôle essentiel dans la compaction finale de l'enzyme sous sa forme native.L'échange H/D a permis de caractériser un intermédiaire de dépliement, le globule fondu, comme ayant une structure très fluctuante. Il a aussi permis de montrer des changements structuraux importants de la CK-MM permettant un alignement des substrats lors de la formation du complexe analogue de l'état de transition.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Redox signaling : insights into the interaction mechanism of sulfiredoxin with peroxiredoxin and ATP

International audienc

Studing redox enzymes interactions at the single-molecule level. Restitution des PEPS Mirabelle

Restitution des PEPS Mirabell

Interactions between enzymes of the intracellular redox signaling

International audienc

Hydrogen/deuterium exchange studies of native rabbit MM-CK dynamics

Creatine kinase (CK) isoenzymes catalyse the reversible transfer of a phosphoryl group from ATP onto creatine. This reaction plays a very important role in the regulation of intracellular ATP concentrations in excitable tissues. CK isoenzymes are highly resistant to proteases in native conditions. To appreciate localized backbone dynamics, kinetics of amide hydrogen exchange with deuterium was measured by pulse-labeling the dimeric cytosolic muscle CK isoenzyme. Upon exchange, the protein was digested with pepsin, and the deuterium content of the resulting peptides was determined by liquid chromatography coupled to mass spectrometry (MS). The deuteration kinetics of 47 peptides identified by MS/MS and covering 96% of the CK backbone were analyzed. Four deuteration patterns have been recognized: The less deuterated peptides are located in the saddle-shaped core of CK, whereas most of the highly deuterated peptides are close to the surface and located around the entrance to the active site. Their exchange kinetics are discussed by comparison with the known secondary and tertiary structures of CK with the goal to reveal the conformational dynamics of the protein. Some of the observed dynamic motions may be linked to the conformational changes associated with substrate binding and catalytic mechanism

Insights into Peroxiredoxin-Sulfiredoxin interaction mechanism at multiscale levels

International audienc

Mechanism and substrate specificities of bacterial and human thiosulfate sulfurtransferase: Human TSTD1 displays dual physiological roles in hydrogen sulfide production and elimination

International audienc

Evidence That Glutathione and the Glutathione System Efficiently Recycle 1-Cys Sulfiredoxin In Vivo

International audienceAims: Typical 2-Cys peroxiredoxins (2-Cys Prxs) are Cys peroxidases that undergo inactivation by hyperoxidation of the catalytic Cys, a modification reversed by ATP-dependent reduction by sulfiredoxin (Srx). Such an attribute is thought to provide regulation of 2-Cys Prxs functions. The initial steps of the Srx catalytic mechanism lead to a Prx/Srx thiolsulfinate intermediate that must be reduced to regenerate Srx. In Saccharomyces cerevisiae Srx, the thiolsulfinate is resolved by an extra Cys (Cys48) that is absent in mammalian, plant, and cyanobacteria Srxs (1-Cys Srxs). We have addressed the mechanism of reduction of 1-Cys Srxs using S. cerevisiae Srx mutants lacking Cys48 as a model. Results: We have tested the recycling of Srx by glutathione (GSH) by a combination of in vitro steady-state and single-turnover kinetic analyses, using enzymatic coupled assays, Prx fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reverse-phase chromatography coupled to mass spectrometry. We demonstrate that GSH reacts directly with the thiolsulfinate intermediate, by following saturation kinetics with an apparent dissociation constant of 34 mu M, while producing S-glutathionylated Srx as a catalytic intermediate which is efficiently reduced by the glutaredoxin/glutathione reductase system. Total cellular depletion of GSH impacted the recycling of Srx, confirming in vivo that GSH is the physiologic reducer of 1-Cys Srx. Innovation: Our study suggests that GSH binds to the thiolsulfinate complex, thus allowing non-rate limiting reduction. Such a structural recognition of GSH enables an efficient catalytic reduction, even at very low GSH cellular levels. Conclusion: This study provides both in vitro and in vivo evidence of the role of GSH as the primary reducer of 1-Cys Srxs. Antioxid. Redox Signal. 22, 731-743

Catalytic properties of a bacterial acylating acetaldehyde dehydrogenase: evidence for several active oligomeric states and coenzyme A activation upon binding

International audienceUntil the last decade, two unrelated aldehyde dehydrogenase (ALDH) superfamilies, i.e. the phosphorylating and non-phosphorylating superfamilies, were known to catalyze the oxidation of aldehydes to activated or non-activated acids. However, a third one was discovered by the crystal structure of a bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase/acylating acetaldehyde dehydrogenase (DmpFG) from Pseudomonas sp. strain CF600 (Manjasetty et al., Proc. Natl. Acad. Sci. USA 100 (2003) 6992-6997). Indeed, DmpF exhibits a non-phosphorylating CoA-dependent ALDH activity, but is structurally related to the phosphorylating superfamily. In this study, we undertook the characterization of the catalytic and structural properties of MhpEF from Escherichia coli, an ortholog of DmpFG in which MhpF converts acetaldehyde, produced by the cleavage of 4-hydroxy-2-ketovalerate by MhpE, into acetyl-CoA. The kinetic data obtained under steady-state and pre-steady-state conditions show that the aldehyde dehydrogenase, MhpF, is active as a monomer, a unique feature relative to the phosphorylating and non-phosphorylating ALDH superfamilies. Our results also reveal that the catalytic properties of MhpF are not dependent on its oligomeric state, supporting the hypothesis of a structurally and catalytically independent entity. Moreover, the transthioesterification is shown to be rate-limiting and, when compared with a chemical model, its catalytic efficiency is increased 10(4)-fold. Therefore, CoA binding to MhpF increases its reactivity and optimizes its positioning relative to the thioacylenzyme intermediate, thus enabling the formation of an efficient deacylation complex