Shikimate dehydrogenase from pepper (Capsicum annuum) seedlings. Purification and properties

Shikimate dehydrogenase (SKDH, EC 1.1.1.25) was extracted from seedlings of pepper (Capsicum annuum L.) and purified 347‐fold. The purification procedure included precipitation with ammonium sulphate and chromatography in columns of Reactive Red‐agarose, Q‐Sepharose and Sephadex G‐100. Pepper SKDH isozymes are separable only using PAGE. The purified enzyme has a relative molecular mass of 67 000 as estimated by gel filtration. The optimum pH of enzyme activity is 10.5 and the optimum temperature is 50°C, but the enzyme is quickly inactivated at temperatures higher than 40°C. The purified enzyme exhibited typical Michaelis‐Menten kinetics and Km values are 0.087 mM for shikimic acid and 0.017 mM for NADP. The mechanism of reaction is sequential considering NADP as a cosubstrate. Ions such as Ca2+, Mg2+ and Mn2+ activate the enzyme, but Zn2+ and Cu2+ are strong inhibitors. Some phenolic compounds such as guaiacol, protocatechuic acid and 2,4‐D are competitive inhibitors of pepper SKDH, showing Ki values of 0.38 mM, 0.27 mM and 0.16 mM, respectively.