Shikimate dehydrogenase from pepper (Capsicum annuum) seedlings. Purification and properties |
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Authors: | José Díaz Fuencisla Merino |
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Institution: | Depto de Bioloxia Animal e Bioloxía Vexetal, Univ. da Coruña, Campus da Zapateira s/n, E-15071 A Coruña, Spain. |
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Abstract: | 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 m M for shikimic acid and 0.017 m M 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 m M , 0.27 m M and 0.16 m M , respectively. |
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Keywords: | Capsicum annuum pepper purification shikimate dehydrogenase Solanaceae |
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