Mechanism of phenylalanine regulation of phenylalanine hydroxylase.

Pterin-Dependent Amino Acid Hydroxylases

“…This loss of activator was concomitant with the loss of activity, as would be expected for relaxation of the R state back to the inactive (≤3%) T state. By using characteristic changes in fluorescence as an assay, l -Phe was found to inhibit this relaxation process, i.e., the R-state enzyme is “kinetically trapped” by its activator [ l -Phe]) and reverse reactions have the same, pronounced temperature dependence that corresponds to an Arrhenius activation barrier of ∼34 kcal (mol subunit) -1 . , This correctly predicts that the activation equilibrium ( K d,app for the allosteric l -Phe site) has a shallow temperature dependence, implying the existence of a small Δ H associated with allosteric activation.…”

“…Upon irradiation, this class of compounds dissociates N 2 , leaving a highly reactive nitrene center poised on the cofactor near the enzyme site of interest. Rat liver PAH was inactivated by irradiation with 302 nm light in the presence of ANBADP (or to a lesser extent, in its absence, since PAH is mildly photolabile) . This compound competitively inhibited PAH vs DMPH 4 ( K I = 9 μM); photoinactivation of PAH by ANBADP was partially blocked by 6MPH 4 or the effective competitive inhibitor 5-deazapterin.…”

“…No posttranslational modifications other than phosphorylation are known, , and even this causes only subtle in vitro kinetic changes. A procedure involving a l -Phe-mediated hydrophobic affinity step was introduced by Shiman in 1979, and has proven to be a versatile method for purifying high activity PAH from a variety of other natural (human, mouse, cow, baboon, rabbit, and goose) and recombinant (rat gene, E. coli , or baculovirus − ) sources. (Different methods have been used to purify monkey PAH. , ) This method of purification is easily scaled up and gives consistent, high activity yields of PAH; as a result it is essentially the only procedure now used, although various other procedures have been employed. ,,− Recombinant sources of PAH are as active as the enzyme isolated from hepatic tissue, contain up to one equivalent of tightly bound non-heme iron per subunit, and lack phosphate.…”

“…The rate of activation was shown to depend on the l -Phe concentration and pH . The rate of loss of labeled amino acid from R-state subunits was further investigated in a particularly elegant study that demonstrated l -[ 14 C]Phe bound to the allosteric activation site is protected from hydroxylation …”

Missense Mutations in the N-Terminal Domain of Human Phenylalanine Hydroxylase Interfere with Binding of Regulatory Phenylalanine

“…These findings suggest that binding of phenylalanine to the regulatory domain of PAH induces conformational changes that displace the autoregulatory sequence from its position at the active site. Activation of PAH is a cooperative, reversible process involving all three functional domains and all four subunits in the holoenzyme (Shiman et al 1990;Kaufman 1993;Davis et al 1997).…”