Kinetics of Phenylalanine Hydroxylase with Analogs of Tetrahydrobiopterin

Phenylalanine hydroxylase catalyzes the conversion of phenylalanine to tyrosine using molecular oxygen and a tetrareduced pteridine, tetrahydrobiopterin. In order to clarify the mode of interaction of tetrahydrobiopterin in this hydroxylation, the effects of ten differently substituted tetra-hydropteridines on the action of partially purified rat liver phenylalanine hydroxylase were determined. Activity was assayed (1) by a specific spectrophotometric assay in which the phenylalanine-dependent oxidation of the pteridine co-factor is monitored at 330 nm (Ayling, J. E., Pirson, R., Pirson, W., and Boehm, G. (1973), Anal. Biochem. 51, 80), and (2) from the amount of tyrosine formed in the presence of dithiothreitol as a cofactor regenerating system. No activity or inhibition was found with any nonreduced pteridines, or with 2,4-diketo-6,7-dimethyltetrahydropteridine (H₄Pt), 2-amino-4-keto-6-carboxyl-H₄Pt, or tetrahydroaminopterin. Four of the tetrareduced pteridines were inhibitory. In order of increasing inhibitory potency these were 4-amino-, 4-keto-, 4-keto-6,7-dimethyl-, and 2-amino-4-keto-6,7-diphenyl-H₄Pt. The inhibition with respect to cofactor was mixed for the first two and competitive for the last two inhibitors. All showed mixed inhibition with respect to phenylalanine, except 4-keto-6,7-dimethyl-H4Pt, which was noncompetitive. Tetrahydropteridines active as cofactors were 2-amino-4-keto-, 2-amino-4-keto-6,7-dimethyl-, 2,4-diamino-6,7-dimethyl-H₄Pt, and tetrahydrobiopterin, in order of increasing apparent maximum velocity. Except for 2-amino-4-keto-H₄Pt, each reacted stoichiometrically with phenylalanine to form tyrosine. With 2-amino-4-keto-H₄Pt only about half the predicted amount of tyrosine was formed. These results indicate that a 2-amino group, or possibly a similar electron donor, is essential for cofactor activity. At the 4 position a keto or an amino group produces an equally effective cofactor. The affinity of tetrahydropteridines for phenylalanine hydroxylase is affected by the substituent at the 6 position. With a methyl or a hydrogen at this position the affinity is less than that of tetrahydrobiopterin, which has a dihydroxypropyl, and with a carboxyl or p-aminobenzoylglutamate (as in aminopterin) there is no binding at all. In addition, a substituent at the 6 position appears to be necessary for tetrahydropteridines to bind in a specific manner at the active site, since in the absence of a 6 substituent inhibition is mixed, rather than competitive, with inhibitory compounds, and for pteridines with cofactor activity the normal stoichiometry is not observed. The inhibition observed with 2-amino-4-keto-6,7-diphenyl-H₄Pt suggests that the position of phenylalanine in the complex is adjacent to the tetrahydrobiopterin 7 position.