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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 N 4.3 feature is inversely proportional to the specific activity of the enzyme, whereas the intensity of the geff E 6.7-5.4 region correlates with the activity of the enzyme. The I n our attempts to understand the mechanism of action of phenylalanine hydroxylase (PAH) from rat liver, we (Gottschall et al., 1982) recently confirmed and extended an earlier observation by Fisher et al. (1972) that a tightly bound non-heme iron was necessary for the activity of the enzyme. We demonstrated that (1) the correct stoichiometry is one atom per subunit, (2) the enzymatic activity is proportional to the iron content, and (3) the iron can be removed from the enzyme and restored with nearly complete recovery of initial activity. In this paper we corroborate and expand the independent discovery (Marota & Shiman, 1984) that PAH is initially reduced to the catalytically active enzyme by the concomitant oxidation of 6-methyltetrahydropterin (6MPH4) directly to the quinonoid form in a stoichiometric reaction requiring oxygen but without the formation of reduced forms of oxygen such as superoxide and hydrogen peroxide, or of tyrosine. In this paper we (1) propose a two-step kinetic sequence for PAH activation and its catalytic turnover based on stopped-flow kinetic and complimentary product data, (2) define by EPR that the resting state of the activatable enzyme is associated with signals observed at geff = 6.7 and 5.4 consistent with high-spin Fe3+ (S = (3) show that the additional EPR signal observed at geff = 4.3 is associated with a form of the enzyme that is incapable of turnover, and (4) link the prereduction step to the conversion of PAH from an Fe3+ to an Fez+ state. Finally, we demonstrate that dithionite can substitute for 6MPH4 in the prereduction step and that the addition of one electron/subunit is sufficient to impart tightly coupled turnover. Experimental Procedures MaterialsDoubly distilled deionized water was used throughout. All reagents were of the highest grade commercially available. 0006-2960/84/0423-1295%01 SO10latter features are lost upon addition of phenylalanine under anaerobic or aerobic conditions. In the presence of ophenanthroline, the operation of the prereduction step results in nearly quantitative trapping of the iron in an Fez+ redox state. Dithionite can substitute for 6-methyltetrahydropterin in an anaerobic prereduction step, generating a catalytically active phenylalanine hydroxylase containing ...

Iron can be bound to phenylalanine hydroxylase (PAH) in two environments. The assignment of the electron paramagnetic resonance spectrum of PAH to two, overlapping high-spin ferric signals is confirmed by computer simulation. Both environments are shown to be populated in the crude enzyme. Reconstitution of the apoenzyme demonstrated that the two iron environments are not interconvertible. Oxygen consumption during PAH reduction by tetrahydropterin in the absence of phenylalanine but not in its presence explains the different reduction stoichiometries (tetrahydropterin:enzyme) that have been observed.

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[25,28,66]) are thus both dispensable for growth on glucose, but the cell requires one or the other for growth on fructose. The two hexokinases are not functionally interchangeable, however, as there are differences in the phenotypes of null mutations in the two genes, notably in the growth rate on glucose and the degree of remaining glucose repression (47).Yeast hexokinase isozymes have been the subject of a considerable number of biochemical studies (for reviews, see references 17 and 59). The in vitro properties of the purified isoenzymes have been characterized previously (20)(21)(22)(69)(70)(71). Chemical modification studies demonstrated that a thiol group (55), a glutamyl residue (58), and at least one arginyl residue (11,57) are essential for the catalytic activity of yeast hexokinase; these residues have not been specifically identified. The pH dependence of hexokinase catalytic activity (72) indicates that an acidic group is crucial for catalysis, further supporting a role for a glutamyl residue in catalysis.The yeast hexokinases have also been studied extensively by X-ray crystallography. The three-dimensional structures of the hexokinase II monomer in a complex with the glucose analog O-toluoylglucosamine (67), its dimer without substrate (4-6), and hexokinase I with glucose (8, 9) have been * Corresponding author.