Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus.
Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 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 mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight 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 prephenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.
D000421:Alcaligenes / Q000201:enzymology*; D000645:Ammonium Sulfate; D002474:Cell-Free System; D005591:Chemical Fractionation; D002826:Chorismate Mutase / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography; D006836:Hydro-Lyases / Q000302:isolation & purification; D007535:Isomerases / Q000302:isolation & purification; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification; D010649:Phenylalanine / Q000096:biosynthesis; D010667:Phenylpyruvic Acids / Q000378:metabolism; D011302:Prephenate Dehydratase / Q000302:isolation & purification / Q000378:metabolism; D011303:Prephenate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014443:Tyrosine / Q000096:biosynthesis
D008670:Metals; D010667:Phenylpyruvic Acids; D013439:Sulfhydryl Reagents; D014443:Tyrosine; D010649:Phenylalanine; D010088:Oxidoreductases; D011303:Prephenate Dehydrogenase; D006836:Hydro-Lyases; D011302:Prephenate Dehydratase; D007535:Isomerases; D002826:Chorismate Mutase; D000645:Ammonium Sulfate