Spectroscopic and kinetic properties of unphosphorylated rat hepatic phenylalanine hydroxylase expressed in Escherichia coli - Comparison of resting and activated states

The non-heme iron-dependent metalloenzyme, rat hepatic phenylalanine hydroxylase (EC 1.14.16.1; phenylalanine 4 monooxygenase (PAH)) was overexpressed in Escherichia coli and purified to homogeneity, allowing a detailed comparison of the kinetic, hydrodynamic, and spectroscopic properties of its allosteric states, The homotetrameric recombinant enzyme, which is highly active and contains 0.7-0.8 iron atoms per subunit, is identical to the native enzyme in several properties: K-m, 6-methyltetrahydropterin = 61 mu M and L-Phe = 170 mu M; V-max = 9 s(-1) (compared to 45 mu M, 180 mu M, and 13 s(-1) for the rat hepatic enzyme). L-Phe and lysolecithin treat ment induce the rPAH(T) --> rPAH(R) (where r is recombinant) allosteric transformation necessary for rPAH activity, Characteristic changes in the fluorescence spectra, increased hydrophobicity, a large activation energy barrier, and a 10% volume increase of the tetrameric structure are consistent with a significant reorganization of the protein following allosteric activation, However, optical and EPR spectroscopic data suggest that only minor changes occur in the primary coordination sphere (carboxylate/histidine/water) of the catalytic iron center, Detailed steady state kinetic investigations, using 6-methyltetrahydropterin as cofactor and lysolecithin as activator, indicate rPAH follows a sequential mechanism. A catalytic Arrhenius E(act) of 14.6 +/- 0.3 kcal/mol subunit was determined from temperature dependent stopped-flow kinetics data, rPAH inactivates during L-Phe hydroxylation with a half-life of 4.3 min at 25 degrees C, corresponding to an Arrhenius E(act) of 10 +/- 1 kcal/mol subunit for the inactivation process, Catechol binding (2.4 x 10(6) M(-1)) is shown to occur only at catalytically competent iron sites, Ferrous rPAH binds NO, giving rise to an S-T = 3/2 spin system.