The non-enzymic hydroxylation of phenylalanine to tyrosine by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine

Abstract

1. Phenylalanine is converted into tyrosine by incubation in air with 6,7-dimethyltetrahydropterin, which is a cofactor for the enzymic hydroxylation. This can cause serious inaccuracies in assays of phenylalanine hydroxylase. 2. The non-enzymic reaction is not specific for l-phenylalanine. 3. m-Tyrosine, o-tyrosine and dihydroxyphenylalanines are formed in addition to p-tyrosine; their chromatographic separation and assay are described. 4. l-[(14)C]Phenylalanine as purchased or soon after purification contains p- and m-tyrosine, both of which can cause errors in the assay of phenylalanine hydroxylase. 5. Catalase prevents the non-enzymic hydroxylation. Thiol compounds in low concentrations stimulate the reaction but in high concentrations are inhibitory. Fe(2+) and metal complexing agents have small stimulatory effects. 6. The mechanism of the non-enzymic reaction and its possible relation to the enzymic hydroxylation of phenylalanine are discussed; it is suggested that phenylalanine is attacked by a peroxide of the cofactor.

Phenylalanine as substrate for tyrosine hydroxylase in bovine adrenal chromaffin cells

“…to hydroxylate phenylalanine has been reported previously [1][2][3][4][5], but for some reason the results on partially purified tyrosine hydroxylase [1][2][3][4] have not been widely accepted, perhaps because of reports on non-enzymic conversion of phenylalanine into tyrosine under certain reaction conditions [6,7], as well as enzymically catalysed formation of some m-tyrosine, which was shown not to be a substrate for the enzyme [8]. Reports on tyrosine hydroxylase isolated from rat pheochromocytoma (PC12) cells by two separate groups claimed that phenylalanine was not hydroxylated by their enzyme preparations [10,11], even under conditions in which 'one turnover event per enzyme molecule could be detected' [10].…”

“…Direct injection of labelled phenylalanine into brains of rats resulted in the formation of labelled catecholamines [5]. In spite ofthese early observations, the possibility that phenylalanine could be a physiologically significant precursor for catecholamine biosynthesis in neurons and chromaffin cells has remained controversial, probably for the following reasons: (1) nonenzymic oxidation of phenylalanine to tyrosine in the presence of cofactor and dioxygen is known to occur [6,7]; (2) one of the products of the enzymic reaction catalysed by tyrosine hydroxylase was claimed to be m-tyrosine [8], which was found not to be a substrate for the enzyme [9]; and (3) highly purified preparations of tyrosine hydroxylase were reported not to catalyse phenylalanine hydroxylation [10,1 1]. In our laboratory, however, phenylalanine was found to be almost as good a substrate for highly purified tyrosine hydroxylase as tyrosine itself under certain experimental conditions in vitro [12].…”

Incorporation of Ortho- and Meta-Tyrosine Into Cellular Proteins Leads to Erythropoietin-Resistance in an Erythroid Cell Line

“…When excessive amount of hydroxyl radical is present, L-phenylalanine is converted into meta-tyrosine and ortho-tyrosi ne, besides the enzymatic formation of the physiological isomer, para-tyrosine [15]. Gurer-Orhan et al proved that the concentration-dependent integration of meta-tyrosine into cellular proteins may be a mechanism of cytotoxicity [16].…”

Isolation and characterization of dihydropteridine reductase from Pseudomonas species

“…The increase in pterin-catalyzed non-enzymatic NADH oxidation at low pH values was probably not observed by Nielsen et al (24) since they used a double-beam spectrophotometer. It is likely, based on several previous reports (14,28), that a peroxide derivative of tetrahydropterin is formed. The reduction of this product by NADH, a reaction usually catalyzed by dihydropteridine reductase, would account for the rapid NADH oxidation we observe.…”