Reductive activation of phenylalanine hydroxylase and its effect on the redox state of the non-heme iron.

Phenylalanine hydroxylase undergoes an obligatory prereduction step in order to become catalytically active as shown by stopped-flow kinetics and by measuring tyrosine formation at limiting 6-methyltetrahydropterin levels. This initial step requires oxygen and involves conversion of 6-methyltetrahydropterin directly to the quinonoid form with or without phenylalanine. The EPR spectrum of the resting enzyme (geff = 9.4-8.7, 4.3 and geff = 6.7, 5.4) is consistent with two species possessing distinctively different ligand environments for the non-heme, high-spin Fe3+. The intensity of the geff congruent to 4.3 feature is inversely proportional to the specific activity of the enzyme, whereas the intensity of the geff congruent to 6.7-5.4 region correlates with the activity of the enzyme. The latter features are lost upon addition of phenylalanine under anaerobic or aerobic conditions. In the presence of o-phenanthroline, the operation of the prereduction step results in nearly quantitative trapping of the iron in an Fe2+ redox state. Dithionite can substitute for 6-methyltetrahydropterin in an anaerobic prereduction step, generating a catalytically active phenylalanine hydroxylase containing Fe2+ that functions aerobically to produce tyrosine from added 6-methyltetrahydropterin in a 1/1 stoichiometry. Reductive titration of the hydroxylase by dithionite is consistent with the addition of one electron/subunit for coupled turnover. The implications of these findings for the mechanism of action of this enzyme are briefly discussed.

The mechanism of phenylalanine hydroxylase.

The site of oxygen binding during phenylalanine hydroxylase (PAH)-catalyzed turnover of phenylalanine to tyrosine has been tentatively identified as the 4a position of the tetrahydropterin cofactor, based on the spectral characteristics of an intermediate generated from both 6-methyltetrahydropterin and tetrahydrobiopterin during turnover. The rates of appearance of the intermediate and tyrosine are equal. Both rates exhibit the same dependence on enzyme concentration. PAH also requires 1.0 iron per 50,000-dalton subunit for maximal activity. A direct correlation between iron content and specific activity has been demonstrated. Apoenzyme can be reactivated by addition of Fe(II) aerobically or Fe(III) anaerobically and can be repurified to give apparently native protein. Evidence from electron paramagnetic resonance implicates the presence of high spin (5/2) Fe(III). As a working hypothesis we postulate that a key complex at the active site may be one containing iron in close proximity to a 4a-peroxytetrahydropterin.

On the mechanism of action of phenylalanine hydroxylase.

The oxidation of 6-methyltetrahydropterin and tetrahydrobiopterin coupled to the formation of tyrosine by phenylalanine hydroxylase generates a precursor species to the quinonoid product that is tentatively identified as a 4a-hydroxy adduct based on its spectral similarity to the 4a-hydroxy-6-methyl-5-deazatetrahydropterin. The rate of appearance of this intermediate and that of tyrosine are equal and hydroxylase catalyzed in accord with the completion of the hydroxylation event. This observation, which confirms and extends an earlier one by Kaufman [Kaufman, S. (1975) in Chemistry and Biology of Pteridines (Pfleiderer, W., Ed.) p 291, Walter de Gruyter, Berlin], serves to link the reaction courses followed by pterin and pyrimidine cofactor analogues and supports the hypothesis that the 4a position is a site of O2 attachment. Thus, as expected, no prereduction of the enzyme was observed in anaerobic experiments utilizing stoichiometric amounts of enzyme and tetrahydropterin in the presence or absence of 1 mM phenylalanine. Activation of the hydroxylase by 1 mM lysolecithin leads to oxidation of the tetrahydropterin in the absence of phenylalanine. A ring-opened pyrimidine analogue of the tetrahydropterin, 2,5-diamino-4-[(meso-1-methyl-2-aminopropyl)amino]-6-hydroxypyrimidine, was studied to examine the possibility of tetrahydropterin ring opening in the enzymatic reaction prior to 4a-hydroxy adduct formation. However, no hydroxylase-catalyzed ring closure was observed.