Kinetic and binding equilibrium studies of dihydroflavonol 4-reductase from Vitis vinifera and its unusually strong substrate inhibition
| Autor: | Bernard Gallois, Béatrice Langlois d'Estaintot, Nadia Trabelsi, Jean Chaudiere, Gilles Sigaud |
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| Přispěvatelé: | Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physiologie Moléculaire des Plantes, Centre de recherches Paul Pascal (CRPP), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
0106 biological sciences
chemistry.chemical_classification 0303 health sciences Isothermal Titration Calorimetry Stereochemistry [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph] Substrate (chemistry) Isothermal titration calorimetry Dehydrogenase Steady-State Kinetics Hummel and Dreyer Technique 01 natural sciences Non-productive Enzyme Substrate Complex 03 medical and health sciences Enzyme chemistry Ionic strength NADPH binding Titration Ternary complex Substrate Inhibition 030304 developmental biology 010606 plant biology & botany |
| Zdroj: | Journal of Biophysical Journal Journal of Biophysical Journal, 2011, 2 (3), pp. 332-344. ⟨10.4236/jbpc.2011.23038⟩ |
| Popis: | 13 pages; International audience; Dihydroflavonol 4-reductase (DFR), a member of the short-chain dehydrogenase family, catalyzes the last common step in the biosynthesis of flavan-3-ols and condensed tannins. Initial rates of DFR were measured by monitoring the 340- nm absorbance decrease resulting from the joint consumption of dihydroquercetin (DHQ) and NADPH, as a function of pH, temperature and ionic strength. At pH 6.5 and 30°C, substrate inhibition was observed above 30 μM DHQ. At lower/non-inhibitory DHQ concentrations, NADP+ behaves as a competitive inhibitor with respect to NADPH and as a mixed inhibitor with respect to DHQ, which supports a sequential ordered mechanism, with NADPH binding first and NADP+ released last. Binding-equilibrium data obtained by means of the chromatographic method of Hummel and Dreyer at pH 7.5 and by isothermal calorimetric titration at pH 6.5 led to the conclusion that ligands of the apoenzyme included NADPH, NADP+ and DHQ. The mecha-nism which best accounts for substrate inhibi-tion at pH 6.5 in the absence of product involves the formation of a binary non-productive E.DHQ complex. Thus, a productive ternary complex cannot be formed when DHQ binds first. This mechanism of inhibition may prevent the ac-cumulation of unstable leucoanthocyanidins within cells. |
| Databáze: | OpenAIRE |
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