Purification and characterization of bifunctional dehydroquinase-shikimate: NADP oxidoreductase from pea seedlings |
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| Affiliation: | 1. Department of Pharmacology, School of Medicine, Faculty of Health Sciences, University of Thessaly, Larissa, Greece;2. Laboratory of Chemistry, Department of Food Science and Human Nutrition, School of Food Biotechnology and Development, Agricultural University of Athens, Athens, Greece;1. Department of Food Quality and Nutrition, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele all’Adige, Trento, Italy;2. Unit of Computational Biology, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 San Michele all’Adige, Trento, Italy;3. Instituto de la Grasa (CSIC), Campus Universidad Pablo de Olavide-Edificio 46, Ctra. de Utrera, km. 1, 41013, Sevilla, Spain;1. Department of Neuroscience, Feinberg School of Medicine, Northwestern University, 303 E Chicago Avenue, Chicago, IL 60611, USA |
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| Abstract: | The bifunctional enzyme dehydroquinase (DHQase, EC 4.2.1.10)-shikimate: NADP oxidoreductase (SHORase, EC 1.1.1.25) has been purified 6500-fold to homogeneity from Pisum sativum shoot tissue. A rapid purification procedure using high performance liquid chromatography was used to isolate the enzyme from chloroplast preparations. The purified enzyme is monomeric with Mr 59 000. Chromatofocusing separates three isoenzymes, two of which are chloroplastic. DHQase and SHORase (forward reaction) show pH optima at pH 7 and apparent Km values of 2.7 x 10−5 M (dehydroquinate), 2.1 x 10−4 M (dehydroshikimate) and 1.5 x 10−5 M (NADPH). Chloride is a competitive inhibitor of DHQase. The SHORase reaction has an ordered (sequential) kinetic mechanism and is unaffected by the presence of DHQ. |
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