Purification and characterization of bifunctional dehydroquinase-shikimate: NADP oxidoreductase from pea seedlings

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 M r 59 000. Chromatofocusing separates three isoenzymes, two of which are chloroplastic. DHQase and SHORase (forward reaction) show pH optima at pH 7 and apparent K m 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.