REGULATION OF RAT-LIVER PHENYLALANINE-HYDROXYLASE .1. KINETIC-PROPERTIES OF THE ENZYMES IRON AND ENZYME REDUCTION SITE

Tetrahydropterins react with phenylalanine hydroxylase at a redox site , a regulatory site, and the catalytic site, but neither the propertie s of nor relationships among these sites are well understood. We have studied the redox site using the fluorescent iron chelators 2,3-dihydr oxynaphthalene and bathophenanthroline; these compounds act as site-sp ecific reporter groups for reactions on oxidized and reduced enzyme, r espectively. The chelators bind reversibly and specifically to the enz yme's iron with 1:1 stoichiometry, high affinity (K-d values similar t o 1 nM), and complete quenching of their own fluorescence. The kinetic behavior of these and other iron chelators indicates that the enzyme' s iron is solvent ac cessible and in a hydrophobic pocket of the prote in. Both ferrous and ferric chelators inhibit phenylalanine hydroxylas e activity. Bathophenanthroline inhibits by binding to Fe2+ on reduced , active enzyme. 2,3-Dihydroxynaphthalene inhibits by binding to Fe3on enzyme that is oxidized during catalysis. This oxidation occurs sim ilar to 1/150 enzyme turnovers, and its rate is increased when p-chlor o- or p-fluorophenylalanine is used as the reaction substrate. Studies of the reaction of tetrahydrobiopterin (BH4) at the enzyme's redox si te showed that BH4 reduces the enzyme more slowly than 6-methyltetrahy dropterin under catalytic and non-catalytic conditions. Reduction occu rs at a distinct site whose binding determinants and reaction characte ristics are different from those of the BH4 regulatory or catalytic si tes, and phenylalanine activated enzyme is reduced more rapidly than u nactivated enzyme. In reducing phenylalanine activated enzyme, BH4 don ates one electron/subunit (1/iron atom); the reduction kinetics sugges t a trihydrobiopterin-free radical as a reaction intermediate.