Abstract
Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin (BH(4)) and non-heme iron-dependent enzyme that hydroxylates L-Phe to L-Tyr. The paramagnetic ferric iron at the active site of recombinant human PAH (hPAH) and its midpoint potential at pH 7.25 (E(m)(Fe(III)/Fe(II))) were studied by EPR spectroscopy. Similar EPR spectra were obtained for the tetrameric wild-type (wt-hPAH) and the dimeric truncated hPAH(Gly(103)-Gln(428)) corresponding to the "catalytic domain." A rhombic high spin Fe(III) signal with a g value of 4.3 dominates the EPR spectra at 3.6 K of both enzyme forms. An E(m) = +207 +/- 10 mV was measured for the iron in wt-hPAH, which seems to be adequate for a thermodynamically feasible electron transfer from BH(4) (E(m) (quinonoid-BH(2)/BH(4)) = +174 mV). The broad EPR features from g = 9.7-4.3 in the spectra of the ligand-free enzyme decreased in intensity upon the addition of L-Phe, whereas more axial type signals were observed upon binding of 7,8-dihydrobiopterin (BH(2)), the stable oxidized form of BH(4), and of dopamine. All three ligands induced a decrease in the E(m) value of the iron to +123 +/- 4 mV (L-Phe), +110 +/- 20 mV (BH(2)), and -8 +/- 9 mV (dopamine). On the basis of these data we have calculated that the binding affinities of L-Phe, BH(2), and dopamine decrease by 28-, 47-, and 5040-fold, respectively, for the reduced ferrous form of the enzyme, with respect to the ferric form. Interestingly, an E(m) value comparable with that of the ligand-free, resting form of wt-hPAH, i.e. +191 +/- 11 mV, was measured upon the simultaneous binding of both L-Phe and BH(2), representing an inactive model for the iron environment under turnover conditions. Our findings provide new information on the redox properties of the active site iron relevant for the understanding of the reductive activation of the enzyme and the catalytic mechanism.
Highlights
These recent structural studies have provided further insight into the function of the iron and the pterin in the catalytic reaction of the aromatic amino acid hydroxylases, little is yet known about the details of electron transfer reactions and the catalytic mechanism
EPR Spectra of Recombinant Human Phenylalanine Hydroxylase—Tris has been found to be an inhibitor of the enzyme, competitive to the pterin cofactor (22), and our previous EPR spectroscopic studies on rat Phenylalanine hydroxylase (PAH) demonstrated that Tris, in its base form, induces changes in the active site iron (20)
The iron environment appears to be more homogenous in the recombinant human PAH (hPAH) than previously observed for the hepatic rat and bovine enzymes, which have been isolated through procedures including preincubation of the crude extracts with ferrous ions and DTT (34)
Summary
Expression and Purification of the Wild-type and Truncated Form of hPAH—Expression in Escherichia coli (TB1 cells) of human wild-type PAH (wt-hPAH) and the truncated hPAH(Gly103-Gln428), i.e. ⌬N102/ ⌬C24-hPAH as fusion proteins with maltose-binding protein, purification of the fusion proteins by affinity chromatography on amylose resin, and their cleavage by the restriction protease factor Xa (New England Biolabs) were performed as described (31, 32). For the EPR-monitored redox titrations the samples were prepared in 50 mM Mops buffer, 0.2 M KCl, pH 7.25, and the final enzyme (wt-hPAH) concentration was 100 –120 M subunit. Bound Cu(II), giving rise to characteristic EPR signals around g ϭ 2.0 –2.3, was removed by incubation of the enzyme with 5 mM EDTA followed by three cycles of dilution and concentration in EDTA-free Mops buffer using Centricon 30 microconcentrators (Amicon). This treatment did not result in any significant change in the shape or intensity of the Fe(III) signal around g ϭ 4.3.
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