Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus

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Abstract
Chorismate mutase and prephenate dehydratase from A. eutrophus H 16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on DEAE cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydroxyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The MW of chorismate mutase-prephenate dehydratase varied from 144,000-187,000 due to the 3 different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of MW 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutase-dehydratase by DEAE-cellulose chromatography. Chromatography on DEAE-Sephadex, Sephadex G-200 and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The MW of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate, free prehenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.