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J. Biol. Chem., Vol. 269, Issue 40, 24637-24646, 10, 1994

Regulation of rat liver phenylalanine hydroxylase. I. Kinetic properties of the enzyme's iron and enzyme reduction site

R Shiman, DW Gray and MA Hill
Department of Biochemistry and Molecular Biology, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033.

Tetrahydropterins react with phenylalanine hydroxylase at a redox site, a regulatory site, and the catalytic site, but neither the properties of nor relationships among these sites are well understood. We have studied the redox site using the fluorescent iron chelators 2,3- dihydroxynaphthalene and bathophenanthroline; these compounds act as site-specific reporter groups for reactions on oxidized and reduced enzyme, respectively. The chelators bind reversibly and specifically to the enzyme's iron with 1:1 stoichiometry, high affinity (Kd values approximately 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 accessible and in a hydrophobic pocket of the protein. Both ferrous and ferric chelators inhibit phenylalanine hydroxylase activity. Bathophenanthroline inhibits by binding to Fe2+ on reduced, active enzyme. 2,3-Dihydroxynaphthalene inhibits by binding to Fe3+ on enzyme that is oxidized during catalysis. This oxidation occurs approximately 1/150 enzyme turnovers, and its rate is increased when p-chloro- or p-fluorophenylalanine is used as the reaction substrate. Studies of the reaction of tetrahydrobiopterin (BH4) at the enzyme's redox site showed that BH4 reduces the enzyme more slowly than 6-methyltetrahydropterin under catalytic and non-catalytic conditions. Reduction occurs at a distinct site whose binding determinants and reaction characteristics are different from those of the BH4 regulatory or catalytic sites, and phenylalanine-activated enzyme is reduced more rapidly than unactivated enzyme. In reducing phenylalanine activated enzyme, BH4 donates one electron/subunit (1/iron atom); the reduction kinetics suggest a trihydrobiopterin-free radical as a reaction intermediate.
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