Enzymic and nonenzymic dehydration reactions of L-arogenate

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Abstract
L-Arogenate, an immediate precursor of either L-tyrosine, L-phenylalanine, or both in many microorganisms and plants, may undergo 2 types of dehydration reactions that yield products of increased stability. Under acidic conditions, a facile aromatization attended by loss of the C-4 hydroxyl and the C-1 carboxyl moieties results in quantitative conversion to L-phenylalanine. When aromatization was largely prevented by maintaining pH in the range of 7.5-12, a second dehydration reaction occurred in which the alanyl side chain and the carboxyl group at C-1 formed a lactam ring to yield spiro-arogenate. The latter reaction occurs at 100.degree. C, roughly 50% conversion being obtained in 2 h. The product formed from L-arogenate was authentic spiro-arogenate, as demonstrated by high-performance liquid chromatography and TLC identification procedures. Further confirmation was obtained by 1H NMR UV spectroscopy and mass spectrometry. The conversion of L-arogenate to spiro-arogenate is not known to be enzyme catalyzed. The other dehydratase reaction, however, is catalyzed in nature by an enzyme denoted arogenate dehydratase. An improved assay is described for this in which [3H]dansylderivatives of L-arogenate (substrate) and L-phenylalanine (product) are separated by using bidimensional TLC. The radioactive reaction product is then quantitated. This assay was used to study partially purified arogenate dehydratase from Pseudomonas diminuta, an organism that depends upon the arogenate pathway for L-phenylalanine biosynthesis. This enzyme possessed a Km,app of 0.63 mM for L-arogenate and was sensitive to inhibition by L-phenylalanine, 50% inhibition being obtained at 70 .mu.M L-phenylalanine. In contrast, arogenate dehydratase isolated from P. aeruginosa, an organism that possesses both the phenylpyruvate and arogenate routes to L-phenylalanine, was insensitive to inhibition by L-phenylalanine.