The inactivation of phenylalanine hydroxylase by 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and the aerobic oxidation of the latter. The effects of catalase, dithiothreitol and reduced nicotinamide–adenine dinucleotide

Abstract

1. Phenylalanine hydroxylase is inhibited by its cofactor, 6,7-dimethyltetrahydropterin. The rate of inactivation, which is irreversible, increases with the concentration of cofactor. 2. Catalase, in sufficient amount relative to cofactor, prevents this inactivation. More tyrosine is formed in the presence of added catalase. 3. Dithiothreitol in the presence of liver extract also prevents inactivation of the enzyme by the cofactor and stimulates hydroxylation of phenylalanine, probably by protecting the cofactor from oxidation and regenerating it from a dihydropterin reaction product. Dithiothreitol restores linearity of rate at very low enzyme concentrations. 4. Dimethyltetrahydropterin is unstable when the solution is exposed to air but is stabilized by dithiothreitol the aerobic oxidation of which is greatly accelerated by dimethyltetrahydropterin. 5. NADH together with liver extract stabilizes the cofactor but not phenylalanine hydroxylase. 6. It is suggested that either hydrogen peroxide or an organic peroxide formed by oxidation in air of the cofactor is the substance attacking phenylalanine hydroxylase, dithiothreitol and cofactor.

The isolation and properties of phenylalanine hydroxylase from human liver

“…for four separately prepared batches of enzyme). No inhibition was observed, under our assay conditions, even at 1.0mM cofactor concentration (Jakubovic et al, 1971). V,,,,.…”

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

“…o-and m-Tyrosine were purchased from Sigma Chemical Co. (St Louis, Mo., U.S.A.) who assayed them by paper chromatography and N content at >99% and >98% pure respectively. Other materials used were as reported in the preceding paper (Jakubovi6, Woolf & Chan-Henry, 1971).…”

Article Commentary: The contributions of Louis I Woolf to the treatment, early diagnosis and understanding of phenylketonuria

“…Of immediate clinical relevance to PKU at the time of their publication were his relatively interference-free methods for the determination of phenylpyruvic acid in urine (1952); 64 his improvement of La Du and Michael's method for monitoring phenylalanine in serum (1962); 65 and his paper-chromatographic method for detection of o-hydroxyphenylacetic acid (1967). 31 His improvements to the phenylalanine hydroxylase assay 66,67 contributed to basic research on PKU, and on phenylalanine metabolism in general. One of his most notable achievements was the first purification of human phenylalanine hydroxylase, 68 a feat that allowed subsequent investigation of its metal content and other properties.…”