Báo cáo khoa học: Studies on the regulatory properties of the pterin cofactor and dopamine bound at the active site of human phenylalanine hydroxylase pptx

Studies on the regulatory properties of the pterin cofactor and dopamine bound at the active site of human phenylalanine hydroxylase Therese Solstad 1 , Anne J. Stokka 1 , Ole A. Andersen 2 and Torgeir Flatmark 1 1 Department of Biochemistry and Molecular Biology, University of Bergen, Norway; 2 Department of Chemistry, University of Tromsø, Norway The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate ( L -phenylalanine, L-Phe) activation, pterin cofactor inhibition and phos- phorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)- L -erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global con- formational transition (hysteresis) triggered by L-Phe bind- ing, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMP- dependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available three- dimensional crystal structure information on the ligand-free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side-chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L-Phe binding triggers local as well as global conformational changes of the pro- tomer resulting in a displacement of the cofactor bound at the active site by 2.6 A ˚ (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dop- amine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism inclu- ding its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities. Keywords: tetrahydrobiopterin; dopamine; phosphoryl- ation; surface plasmon resonance; regulation. Mammalian phenylalanine hydroxylase (PAH, phenylala- nine 4-monooxygenase, EC 1.14.16.1) catalyses the stereo- specific hydroxylation of L -phenylalanine (L-Phe) to tyrosine (L-Tyr) in the liver [1], kidney [2,3] and melano- cytes [4], utilizing (6R)- L -erythro-5,6,7,8-tetrahydrobiop- terin (H 4 biopterin) as the physiological electron donor. A lack or dysfunction of this enzyme in humans is associated with the autosomal recessive disease hyperphenylalanine- mia/phenylketonuria [5] (http://www.mcgill.ca/pahdb). It has been estimated that the liver contains a sufficiently high level of PAH and pterin cofactor to remove all free L-Phe from the blood within a few minutes if all enzyme molecules are fully active [6]. However, early on it was recognized that the activity of PAH is effectively controlled by several mechanisms in order to maintain the phenylalanine and tyrosine homeostasis in vivo despite great fluctuations in the dietary intake of L-Phe and the overall rate of protein catabolism. It became apparent that the short-term control of rat liver PAH (rPAH) is kinetic, primarily through an activation of the enzyme by L-Phe [7,8]. This activation is a cooperative, reversible process involving all protomers of the 200-kDa enzyme homotetramer [7,8]. rPAH is activated several fold in vitro by preincubation with L-Phe as well as by some amino acid analogues [9]. The recombinant human enzyme (hPAH) has similar regulatory properties in vitro as rPAH, i.e. the tetrameric form binds L-Phe with positive cooperativity with a Hill coefficient (h) of 1.6–1.9, and preincubation with substrate results in a sixfold to eightfold activation of the enzyme [10]. In addition to its catalytic function as an electron donor in the reduction of the Correspondence to T. Flatmark, Department of Biochemistry and Molecular Biology, University of Bergen, A ˚ rstadveien 19, N-5009 Bergen, Norway. Fax: + 47 55586400, Tel.: + 47 55586428, E-mail: torgeir.flatmark@ibmb.uib.no Abbreviations: hPAH, human phenylalanine hydroxylase; rPAH, rat phenylalanine hydroxylase; h, Hill coefficient; H 2 biopterin, (6R)- L -erythro-7,8-dihydrobiopterin; H 4 biopterin, (6R)- L -erythro- 5,6,7,8-tetrahydrobiopterin; H 4 6-methyl-pterin, 6-methyl-5,6,7, 8-tetrahydropterin; IPTG, isopropyl-thio-b- D -galactoside; L-Phe, L -phenylalanine; MBP, maltose binding protein; PKA, cAMP dependent protein kinase; wt, wild-type. Enzyme: phenylalanine 4-monooxygenase or phenylalanine hydroxylase (EC 1.14.16.1). (Received 22 October 2002, revised 15 January 2003, accepted 20 January 2003) Eur. J. Biochem. 270, 981–990 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03471.x catalytic iron and activation of dioxygen, the natural pterin cofactor H 4 biopterin inhibits the substrate activation of rPAHbyformingabinaryenzymeÆH 4 biopterin complex [7,11]. The activity of rPAH and human phenylalanine hydroxylase (hPAH) is also regulated by post-translational mechanisms, notably phosphorylation of Ser16 by cAMP- dependent protein kinase (PKA), which sensitizes the enzyme for activation by L-Phe [12,13]. Whereas the binding of substrate slightly increases the rate of phos- phorylation of rPAH and hPAH by PKA, H 4 biopterin acts as a negative effector on the same process [12,13]. Finally, nonenzymatic deamidation of labile Asn residues in hPAH during its expression in, e.g. Eschericia coli,hasmore recently been shown to result in a threefold increase in its catalytic efficiency [10]. The molecular basis for the inhibitory effects observed for the natural pterin cofactor (H 4 biopterin/H 2 biopterin) has been addressed in a series of studies on rPAH including direct binding measurements [14], steady-state kinetic ana- lysis [11] and modulation of Ser16 phosphorylation by PKA [12]. The binding studies by Shiman and collaborators [11,15] were interpreted to support a working model that includes three types of binding sites for H 4 biopterin. The sites were (a) a redox site, involved in the reduction of the active site iron [Fe(III) fi Fe(II)], (b) a catalytic site, involved in the activation of dioxygen and hydroxylation of L-Phe, and (c) a regulatory site outside the active site, responsible for its inhibitory properties. However, recent crystal structure analyses of the ligand-free, the binary and ternary complexes of hPAH [16–21], as well as the complementary NMR-molecular modelling structural stud- ies [22], have not been able to identify more than a single cofactor binding site, i.e. the binding at the active site, with two alternative orientations in the binary and ternary complex [20,21]. Thus, the molecular basis for the regula- tory (inhibitory) properties of H 4 biopterin is still a matter of debate, both with respect to the domain localization of the inhibitory binding site and the essential importance of its dihydroxypropyl side-chain for inhibition. In the present study, we address these questions as well as the related regulatory properties of the catecholamine inhibitor dop- amine, which is known to bind covalently (bidentate coordination) to the active site iron [17] with high affinity, employing recombinant tetrameric wt-hPAH, with the three-dimensional crystal structural information on this enzyme [16–21] as a reference. Materials and methods Materials The restriction proteases enterokinase and factor Xa were obtained from Invitrogen (The Netherlands) and Protein Engineering Technology (ApS, Aarhus, Denmark), respect- ively. The catalytic subunit of cAMP-dependent protein kinase was purified to homogeneity from bovine heart and was a generous gift from S. O. Døskeland, Department of Anatomy and Cell Biology, University of Bergen. The pterin cofactors (H 4 biopterin, H 2 biopterin and H 4 6-methyl- pterin) were purchased from B. Schircks Laboratory (Joana, Switzerland). Specific chemicals are mentioned in the text elsewhere. Expression and purification of recombinant hPAH The pMAL expression system was used for the production of the wild-type fusion proteins MBP-(D 4 K) ek -hPAH, MBP-(IEGR) Xa -hPAH and its double truncated form MBP-(IEGR) Xa -hPAH(Gly103-Gln428) with maltose binding protein as the fusion partner [23]. Cells were grown at 37 °C, and expression was induced at 28 °Cbythe addition of 1 m M isopropyl-thio-b- D -galactoside (IPTG); the cells were harvested after 2, 8 or 24 h of induction. Full- length hPAH (residues 1–452) was obtained by enterokinase (at D 4 K) or factor Xa (at IEGR) cleavage and hPAH(Gly103–Gln428) by factor Xa cleavage, and the tetrameric (full-length form) and dimeric form (the trun- cated catalytic core enzyme) were isolated by size-exclusion chromatography [23]. Protein measurements The concentration of purified enzyme forms was measured by the absorbance at 280 nm, using the absorption coeffi- cient e 280 ¼ 1.63 for the fusion protein MBP-(pep)-hPAH and 1.0 for the isolated hPAH protein [23]. Protein phosphorylation The tetrameric wt-hPAH was phosphorylated by PKA as described [13]. The standard reaction mixture contained 15 m M Na-Hepes (pH 7.0), 0.1 m M ethylene glycol bis (a-amino ether)-N,N,N¢,N¢-tetraacetic acid, 0.03 m M EDTA, 1m M dithiothreitol, 10 m M magnesium acetate, 60 l M [c- 32 P]-ATP (Amersham, UK), 25 n M of the catalytic subunit of PKA and 4 l M of hPAH. The reaction was performed at 30 °C, and at timed intervals, aliquots of the reaction mixture were spotted on phosphocellulose strips [24] to measure the amount of 32 P transferred to the substrate. In control experiments, the active-site iron in hPAH was prereduced by the noninhibitory [12] H 4 6- methyl-pterin as described [14] prior to initiation of the phosphorylation reaction in the presence of inhibitory H 4 biopterin. Limited proteolysis by trypsin Limited tryptic proteolysis of tetrameric wt-hPAH was performed at 25 °Cin20m M Na-Hepes, 200 m M NaCl at pH 7; the ratio of trypsin to hPAH was 1 : 200 (m/m). Aliquots of 3 lg hPAH were removed at timed intervals and mixed with SDS buffer containing soybean trypsin inhibitor. The protein was finally subjected to SDS/PAGE (10%, w/v), stained with Coomassie Brilliant Blue, and the gels were scanned using DESKSCAN II (Hewlett Packard Co) and further analyzed by using the PHORETIX 1 D analysis software from Nonlinear Dynamics Ltd [10]. Reversed-phase chromatography Reversed-phase chromatography of tryptic peptides was performed using a ConstaMetric Gradient System (Laboratory Data Control, USA) and a 4.6 mm · 10 cm Hypersil ODS C18 column (Hewlett Packard, USA) fitted with a 2-cm guard column. Solvent A was 0.1% 982 T. Solstad et al.(Eur. J. Biochem. 270) Ó FEBS 2003 (w/v) triflouroacetic acid in water, and solvent B was 0.1% trifluoroacetic acid in 70% (v/v) acetonitrile. A linear gradient of 5–100% solvent B at 1 mLÆmin )1 for 60 min was used for the separation of peptides. Absorbance was monitored at 214 nm using a Hewlett- Packard model 1040A photodiode array HPLC detector. Surface plasmon resonance analyses The interactions between tetrameric wt-hPAH and its substrates (L-Phe and H 4 biopterin/H 2 biopterin) were studied by surface plasmon resonance (SPR) analysis using the BiaCore X instrument (BiaCore AB, Uppsala, Sweden). The enzyme, diluted to a final concentration of 0.23 mgÆmL )1 in 10 m M sodium acetate buffer, pH 5.4, was immobilized to the carboxymethylated dextran matrix of a sensor chip (CM5 from BiaCore AB) by the amine coupling procedure [25–27]. The double truncated dimeric form hPAH(Gly103-Gln428) was immobilized to the reference surface by the same procedure [27]. Seventy microlitres of 10 m M dithiothreitol in HBS running buffer (0.15 M NaCl, 3m M EDTA, 0.005% surfactant P20 in 0.01 M Hepes, pH 7.4) was allowed to pass through both flow cells (in series) which empirically decreased the time required to reach a stable baseline [25]. Increasing concentrations of L-Phe diluted in HBS buffer was injected over the immo- bilized enzyme in the absence and presence of 100 l M H 4 biopterin or 500 l M H 2 biopterin. The reduced cofactor (in 1 m M HCl) was kept on ice and the pH adjusted to 7.4 with an accuracy of 0.01 unit immediately before injection [25]. All analyses were carried out at 25 °C and at a constant flow of 5 lLÆmin )1 . The sensorgrams were obtained as the difference in SPR (DRU) response between the sample and the reference cell, which corrects for any changes in the bulk refractive index, together with the small DRU value associated with the binding of the 165-Da substrate. The time-dependent increase in RU (end point at 3 min) was measured directly from the sensorgrams using the cursor guided reading of the X- and Y-coordinates, and was related to the calculated pmol of immobilized enzyme by assuming that 1000 RU corresponds to 1 ng protein boundÆmm )2 [28], i.e. expressed as DRU/(pmol subunitÆmm )2 ). Structural studies The recently reported three-dimensional crystal structures of hPAH and rPAH [16–21] have provided the basis to further explore the molecular mechanism by which the pterin cofactor and dopamine function as negative effectors and how substrate binding completely reverses these effects. The coordinates for the structure of the binary and ternary complexes of the catalytic core domain hPAH(Gly103- Gln428), i.e. of hPAH-Fe(III)ÆH 2 biopterin (PDB id codes 1DMW and 1LRM) [18,20], of hPAH-Fe(II)ÆH 4 biopterin (PDB id code 1J8U) [20] and hPAH-Fe(II)ÆH 4 biopterinÆ 3-(2-thienyl)- L -alanine (PDB id code 1KW0) [21], define the position, orientation, conformation and hydrogen bonding network of the pterin cofactor in the three structures. Superpositions of the binary and ternary complexes of hPAH onto the crystal structure of the ligand-free dimeric rPAH (PDB id code 1PHZ) [19], which contains both the regulatory and the catalytic domains (residues 1–429), were performed to demonstrate the interactions of the dihydroxy- propyl side-chain of the pterin cofactors with the N-terminal autoregulatory sequence. Similarly, the coordinates for the structure of the binary complex with dopamine, i.e. hPAH-Fe(III)Ædopamine (PDB id code 5PAH) [17] define the position and orientation of the inhibitor and the interaction of its main-chain with the autoregulatory sequence. Results Recombinant forms of hPAH were produced at high yields as fusion proteins in E. coli using the pMAL expression vector, and the cleaved tetrameric forms of wt-hPAH and the dimeric truncated form hPAH(Gly103–Gln428) were isolated by size-exclusion chromatography [23]. Aliquots (10 mgÆmL )1 )werestoredinliquidnitrogenandanew aliquot was used for each individual experiment. The conformational differences of tetrameric wt-hPAH resulting from the binding of different ligands (H 4 biopterin, H 2 biop- terin, dopamine and L-Phe) were studied using three different conformational probes, i.e. their effects (a) on the limited proteolysis by trypsin, (b) on the reversible con- formational transition (hysteresis) triggered by substrate binding, as followed in real time by surface plasmon resonance (SPR) analyses, and (c) on the rate of phos- phorylation of Ser16 by PKA. Effect of ligands on the limited proteolysis of recombinant wt-hPAH and its catalytic core Limited proteolysis of rPAH by chymotrypsin has been shown to be a sensitive conformational probe, and among the observed ligand effects was an inhibition of proteolysis by H 4 biopterin [29]. From Fig. 1A it is seen that at saturating concentration (40 l M ), H 4 biopterin has a similar inhibitory effect on the limited proteolysis of tetrameric wt-hPAH by trypsin, which cleaves after Lys/Arg residues in a putative hinge region (residues 111–117, RDKKKNT) connecting the regulatory domain with the catalytic domain [30]. Moreover, the covalently bound inhibitor dopamine (40 l M ) is equally efficient in reducing the susceptibility towards proteolysis. However, when 1 m M L-Phe was also present during the incubation with trypsin, the inhibitory effects of H 4 biopterin and dopamine were completely prevented and the rate of proteolysis increased to approxi- mately the same high level as observed in the presence of substrate alone. By contrast, the dimeric catalytic core enzyme hPAH(Gly103–Gln428) [16], lacking both the N-terminal regulatory domain and the C-terminal tetra- merization domain, was as expected [30] found to be more resistant to proteolysis. When this enzyme form was incubated with either H 4 biopterin, L-Phe, or both ligands combined, no significant effect of the ligands on the rate of proteolysis was observed as compared to the ligand-free enzyme (Fig. 1B). In this case, the results from SDS/PAGE analyses were confirmed by reversed-phase chromatogra- phy of the peptides released during incubation (data not shown). Ó FEBS 2003 Regulatory properties of phenylalanine hydroxylase (Eur. J. Biochem. 270) 983 Effect of H 4 biopterin/H 2 biopterin on the conformational transition (hysteresis) triggered by substrate binding as studied by surface plasmon resonance analysis The effect of the pterin cofactors on the reversible global conformational transition induced by L-Phe binding to tetrameric wt-hPAH was measured in real time by the time- dependent change in refractive index (i.e. as a surface plasmon resonance (SPR) response) of the immobilized enzyme [25–27]. Approximately 25 ngÆmm )2 (0.48 pmol subunitÆmm )2 ) of tetrameric wt-hPAH was immobilized to the dextran matrix of the sample surface, and a slightly lower amount of the dimeric catalytic core enzyme hPAH(Gly103–Gln428) (0.38 pmol subunitÆmm )2 )was immobilized to the reference surface [27]. The time- dependent conformational change (SPR response) of the full-length wt-hPAH was measured as a function of L-Phe concentration and a steady-state (3 min response) binding isotherm was obtained [26,27]. The isotherm observed in the absence of biopterin cofactor was hyperbolic with a concentration of L-Phe at half-maximum saturation ([S] 0.5 )of98±7l M (Fig. 2A,B). Saturation was reached at approximately 2 m M with a DRU-value of 75 RU/(pmol subunitÆmm )2 ). Simultaneous injection of L-Phe (variable concentration) and 100 l M of H 4 biopterin resulted in a  50% decrease in the maximum SPR response to L-Phe (Fig. 2A), and the [S] 0.5 -value for L-Phe increased to 178 ± 11 l M . The presence of 500 l M of the oxidized cofactor H 2 biopterin in the running buffer also lowers the Fig. 2. The effect of pterin cofactor on the global conformational transition of tetrameric wt-hPAH triggered by L-Phe binding as studied by surface plasmon resonance. The effect of increasing L-Phe concen- tration in the absence (d) and presence (s) of the pterin cofactor. (A) 100 l M H 4 biopterin was coinjected with L-Phe. (B) 500 l M of the soluble H 2 biopterin was included in the running buffer. The response in the absence of pterin cofactor represents the average of two separate titration experiments with a basal mean DRU-value of 25090 RU corresponding to 0.12 pmol (0.48 pmol subunit) of immobilized enzyme. The truncated form hPAH(Gly103-Gln428) was present on the reference surface [27]. Fig. 1. The effect of ligand binding on the limited proteolysis by trypsin of full-length wt-hPAH and the truncated form hPAH(Gly103-Gln428). (A) Tetrameric wt-hPAH (24 induction with IPTG) preincubated with either no ligand (d), 40 l M H 4 biopterin (m), 40 l M dopamine (n), 40 l M H 4 biopterin and 1 m M L-Phe (j), 40 l M dopamine and 1 m M L-Phe (h)or1m M L-Phe (s) before being subjected to limited pro- teolysis by trypsin. (B) hPAH(Gly103-Gln428) was preincubated with either no ligand (d), 40 l M H 4 biopterin and 1 m M L-Phe (j)or1m M L-Phe (s) before being subjected to limited proteolysis by trypsin. The ratio hPAH : trypsin was 200 : 1 (m/m). The reactions were allowed to proceed for up to 1 h at 25 °C and aliquots were taken at different time intervals. The reaction was stopped by the addition of soybean trypsin inhibitor (the ratio trypsin : inhibitor 1 : 1.5, m/m) and finally sub- jected to SDS/PAGE stained with Coomassie Brilliant Blue. The gels were scanned using DESKSCAN II (Hewlett Packard Co.); the volume of the bands was analyzed by using the PHORETIX 1 D analysis software from Nonlinear Dynamics Ltd, 1996 [10]. 984 T. Solstad et al.(Eur. J. Biochem. 270) Ó FEBS 2003 maximum SPR response to L-Phe, most significantly observed at concentrations above 200 l M (Fig. 2B), and in this case, the [S] 0.5 -value for L-Phe increased to 123 ± 6 l M . It should be noted that in separate binding experiments with H 4 biopterin alone the [S] 0.5 -value for the cofactor was measured to 5.6 ± 0.8 l M with a D R-value of 25 RU/(pmol subunitÆmm )2 ) at saturation [26]. Effect of active site ligands on the rate of phosphorylation of recombinant hPAH H 4 biopterin and L-Phe have been shown to inhibit and stimulate, respectively, the rate of in vitro phosphorylation of Ser16 by PKA in rPAH at physiologically relevant concentrations [12]. In the present study, the ligand effects on the phosphorylation of Ser16 by PKA in tetrameric wt-hPAH were measured for the reduced cofactor H 4 biop- terin, the oxidized cofactor H 2 biopterin and the catechol- amine inhibitor dopamine. From Fig. 3A it is seen that on preincubation with saturating concentrations (Fig. 3B) of the biopterin cofactors or dopamine the rate of phosphory- lation was decreased, and at 200 l M of the ligands the inhibition was more pronounced for H 4 biopterin ( 26%) and dopamine ( 26%) than for H 2 biopterin ( 12%). The presence of L-Phe during preincubation with H 4 biopterin or dopamine completely reversed their inhibitory effects on phosphorylation (Fig. 3A). From Fig. 3B it is also seen that the most potent inhibitor was dopamine, with a half- maximum inhibition at < 0.1 l M ,andH 4 biopterin ([I ] 0.5 of 1.4 l M , r ¼ 0.96) was more efficient than H 2 biopterin ([I ] 0.5 of 15.8 l M , r ¼ 0.86). The oxidized cofactor reached only a  12% inhibition of phosphorylation compared to the  26% for H 4 biopterin and dopamine. Interestingly, the inhibitory effect of both biopterin cofactors revealed an apparent negative cooperativity, with a Hill coefficient (h)of 0.46 (r ¼ 0.96) for H 4 biopterin (Fig. 3B, insert) and h ¼ 0.75 (r ¼ 0.86) for H 2 biopterin. Hill coefficients < 1 were also observed for the inhibition of the phosphorylation of the tetrameric fusion protein MBP-(pep)-hPAH, and enzyme preparations isolated after a short (2 h) induction period gave reproducibly a higher Hill coefficient with H 4 biopterin than those isolated after a long (24 h) induction period (data not shown). The relative efficiency of the inhibition by the two biopterin cofactors is in good agreement with the previously reported apparent K d values for H 4 biopterin (0.09 ± 0.01 l M )andH 2 biopterin (1.1 ± 0.02 l M ) in their binding to rPAH, as determined by fluorescence quenching titration [14]. The reduction of the active-site iron [Fe(III) fi Fe(II)] by H 4 6-methyl-pterin [14] prior to the phosphorylation assay, did not demonstrate any significant effect on the apparent binding parameters for H 4 biopterin in the present study. Moreover, we have confirmed our previous finding with rPAH as the substrate [12] that the dihydroxypropyl side-chain is required for the cofactor inhibition of phosphorylation (data not shown). The molecular basis for the negative modulatory effects of the pterin cofactor and dopamine binding to wt-hPAH and their reversal by substrate The recently solved high resolution crystal structures of hPAH and rPAH [16–21] have provided a detailed picture of the protein contacts involved in the active site binding of the pterin cofactor [18,20,21], dopamine inhibitor [17] and L-Phe [21]. Thus, the superposition of the hPAH catalytic core structures of the binary complexes with oxidized [18] or reduced [20] pterin cofactor onto the structure of the ligand- free rPAH (containing the regulatory and catalytic domains) [19] revealed that both the reduced and the oxidized cofactor interact with the N-terminal autoregulatory sequence at Ser23. A close-up of this site of interaction (Fig. 4A,B) shows that the O1¢ and O2¢ of the dihydroxypropyl side-chain Fig. 3. The effect of H 4 biopterin, H 2 biopterin, dopamine and L-Phe on the phosphorylation of tetrameric wt-hPAH. (A) Time-course for the phosphorylation of wt-hPAH (4 l M ) at standard incubation condi- tions at 30 °C, including 60 l M [c- 32 P]ATP and 25 n M C-subunit of protein kinase A (PKA). At timed intervals, aliquots of the reaction mixture were spotted on phosphocellulose strips [24] to measure the amount of 32 P transferred to the substrate by scintillation counting. The incubations contained no ligand (d), 40 l M H 4 biopterin (m), 40 l M dopamine (n)or1 m M L-Phe in combination with either 40 l M H 4 biopterin or 40 l M dopamine (h). (B) The effect of increasing concentrations of H 2 biopterin (s), H 4 biopterin (m) and dopamine (n) on the rate of phosphorylation (t ¼ 10 min). Each point in the curves represents the average of four measurements. Insert: a conventional Hill plot on the H 4 biopterin data is shown in the main figure. Y ¼ (v o ) v x )/(v o ) v min ), which is the fractional decrease of phos- phorylation rate seen at the concentration x of H 4 biopterin. v o is the rate in the absence of H 4 biopterin and v min is the rate at very high concentrations of H 4 biopterin. The observed Hill coefficient (h)was found to be 0.46 (r ¼ 0.96). Ó FEBS 2003 Regulatory properties of phenylalanine hydroxylase (Eur. J. Biochem. 270) 985 are sufficiently close to form favourable hydrogen bonds to the carbonyl oxygen of Ser23 (Table 1). However, it is important to note that the dihydroxypropyl side-chain is positioned slightly different in the two redox states of the cofactor and that the distances from Ser23O to O1¢ and O2¢ are slightly different (Table 1) because of the different positions and hybridizations of the C6 atom of the pyrazine ring in the two redox states [18,20]. This diversity of the side- chain position is likely to entail different hydrogen-bonding patterns to Ser23 in the full-length enzyme explaining the redox state dependent regulatory properties of the pterin cofactor. Moreover, the superposition of the ternary struc- ture [21] revealed a similar orientation of the pterin cofactor as in the binary structures [18,20]. However, the reduced cofactor was found to be displaced by 2.6 A ˚ (mean distance) in the direction of the iron and Glu286 upon substrate binding, and in addition, the hydrogen-bonding network for the cofactor was slightly different when compared to the binary structures [18,20]. This displacement of the cofactor results in a loss of stabilizing hydrogen bonds between O1¢ and O2¢ of the dihydroxypropyl side-chain and Ser23O in the autoregulatory sequence (Fig. 4C and Table 1) and thus explaining the complete reversal of the pterin cofactor as a negative effector (Figs 1 and 3A,B). When the crystal structure of the hPAHÆadrenaline/ dopamine binary complex [17] was superimposed onto that of the ligand-free rPAH containing the regulatory and catalytic domains [19] the catecholamine main-chain is also Fig. 4. Stereo view of the site of interaction between the pterin cofactors and dopamine with the regulatory domain. The figure was pro- duced by superimposing the crystal structure of ligand-free dimeric rPAH (PDB id code 1PHZ), which contains both the regulatory and the catalytic domains (residues 1–429) onto the catalytic core crystal structures of (A) binary hPAH with bound H 2 biopterin (PDB id code 1LRM) (B) binary hPAH with bound H 4 biopterin (PDB id code 1J8U) (C) ternary hPAH with bound H 4 biopterin and substrate (PDB id code 1KWO) and (D) binary hPAH with bound dopamine (PDB id code 5PAH). The backbone of rPAH regulatory domain and hPAH catalytic domain are shown in red and green, respectively, while residues Ser23 and Ile25 are shown by ball-and-stick repre- sentation. The figure was prepared using MOLSCRIPT [42]. 986 T. Solstad et al.(Eur. J. Biochem. 270) Ó FEBS 2003 seen to interact with the N-terminal autoregulatory sequence (Fig. 4D and Table 1). The dopamine nitrogen interacts with the carbonyl oxygen of Ser23 and the dopamine Ca atom interacts with the side-chain of Ile25. Discussion The catalytic activity of PAH is regulated by four main mechanisms, i.e. by substrate (L-Phe) activation, biopterin cofactor (H 4 biopterin/H 2 biopterin) inhibition, increased catalytic efficiency on phosphorylation of Ser16 (for review, see [31]) and activation by spontaneous nonenzymatic deamidation of specific labile Asn residues [10]. Internal protein dynamics are intimately connected with these regulatory properties. Based on our previous steady-state enzyme kinetic and phosphorylation studies on rPAH a working model was proposed to explain three of these regulatory properties [12], involving four main conforma- tional states (isomers) of the enzyme. The four conforma- tional states include a ground state for the ligand-free enzyme, an activated state with bound substrate (L-Phe), an inhibited state with bound H 4 biopterin and finally the state of catalytic turnover, i.e. the ternary enzyme-substrate complex. Recent crystal structure analyses of the catalyti- cally active core enzyme in different ligand-bound forms [16–21] and complementary biophysical studies (reviewed in [31]) strongly support such a model. Thus, both H 4 biopterin and L-Phe bind reversibly at the active site of the core enzyme by an induced fit mechanism with defined protein contacts and conformational states [21]. Moreover, PAH is inhibited by the covalent binding of catecholamines, i.e. by bidentate coordination to the active site iron [17]. Whereas the inhibition of the catalytic activity by catecholamines is well understood at the structural level [17], the molecular mechanism of the inhibitory properties of the pterin cofactor (unrelated to its effect as electron donor in the hydroxylation reaction) has been a controversial issue and is further discussed below. On the pterin cofactor binding site Shiman and coworkers [14] have suggested the presence of several putative binding sites for the natural cofactor H 4 biopterin. The proposed binding sites are a regulatory site (outside the active site) that is responsible for the observed inhibitory effects of H 4 biopterin binding and a redox site responsible for the reduction of the active site iron in addition to its binding at the catalytic site as part of a catalytically active ternary complex. Based on the crystal structure of a ligand-free dimeric C-terminal truncated form of rPAH [19] and analogies with the structure of pterin-4a- carbinolamine dehydratase (PCD/DCoH) a putative bind- ing site for H 4 biopterin in the regulatory domain, close to a proposed hinge region (residues 111–117), has been pro- posed [19]. This region is also considered as the target for L-Phe induced proteolytic cleavage by trypsin [30]. More- over, the binding of L-Phe to a second putative site in the regulatory domain was suggested to induce a conforma- tional transition that modifies the intra-subunit interaction between the regulatory and the catalytic domains. This interaction is followed by the formation of a catalytically activated form of the enzyme and an increased susceptibility to tryptic cleavage. Thus, binding of H 4 biopterin to the proposed regulatory site was suggested to prevent the interdomain hinge-bending motion required for activation and susceptibility towards proteolysis [19]. However, recent crystal structure analyses of the binary complex with H 2 biopterin [18] and the binary and ternary complexes with H 4 biopterin [20,21] have defined the protein contacts involved in cofactor binding at the active site and their interactions through the dihydroxypropyl side-chain with the autoregulatory sequence in the regulatory domain (Fig. 4A,B). Thus, no direct structural evidence (by X-ray or NMR) has so far been presented in support of a H 4 biopterin (and L-Phe) binding site in the regulatory domain [18–21,31,32]. Based on this structural information, we here propose an alternative molecular mechanism for the inhibitory effects of the biopterin cofactor on the rate of phosphorylation of Ser16, the limited proteolysis by trypsin and the substrate induced conformational transition (hys- teresis) related to catalytic activation. The inhibited forms of the enzyme with bound biopterin cofactor or dopamine The phosphorylation site in PAH (Ser16) is localized in the N-terminal tail (residues 1–18) of the autoregulatory sequence for which no interpretable electron density has been obtained [19], compatible with a rather flexible structure of the N-terminus [33]. Our experimental data and the crystal structure analysis discussed above, show the interactions of H 4 biopterin, H 2 biopterin and dopamine with the N-terminal autoregulatory sequence at Ser23 and Ile25 (Fig. 4A,B,D; Table 1). We can now present an explanation for the inhibitory effect of the biopterin cofactor and dopamine on the rate of phosphorylation (Fig. 3), on limited proteolysis (Fig. 1A) and on the global conforma- tional transition (hysteresis) related to catalytic activation Table 1. Comparison of distances (A ˚ ) of the superposition of the crystal structure of ligand-free dimeric rPAH (PDB id code 1PHZ), which contains both the regulatory and the catalytic domains (residues 1–429) onto the catalytic core crystal structures of binary hPAH with bound H 4 biopterin (PDB id code 1J8U), binary hPAH with bound H 2 biopterin (PDB id code 1LRM), ternary hPAH with bound H 4 biopterin and substrate (PDB id code 1KW0) and binary hPAH with bound dopamine (PDB id code 5PAH). Distance from Ser23O to H 4 biopterin structure H 2 biopterin structure THA-H 4 biopterin structure Dopamine structure H 4 biopterin O2¢ 2.4 3.8 H 4 biopterin O1¢ 3.0 5.4 H 2 biopterin O2¢ 4.0 H 2 biopterin O1¢ 1.3 Dopamine N1 3.0 Ó FEBS 2003 Regulatory properties of phenylalanine hydroxylase (Eur. J. Biochem. 270) 987 (Fig. 2A,B), i.e. by their direct binding at the active site. That H 4 biopterin is a more potent inhibitor than H 2 biopterin of the global conformational transition triggered by L-Phe binding was most directly demonstrated by the SPR analyses. Whereas 100 l M H 4 biopterin resulted in a  50% reduction in the maximum SPR response to L-Phe binding (Fig. 2A), H 2 biopterin gave only a minor reduction (maximum  11%) at concentrations higher than 200 l M (Fig. 2B). Interestingly, the inhibitory effect of the biopterin cofactor on the rate of phosphorylation revealed an apparent negative cooperativity, with a Hill coefficient (h) of  0.5 for H 4 biopterin (Fig. 3B, insert) and  0.8 for H 2 biopterin. A negative cooperativity has also been reported for the structurally and functionally related human enzyme tyrosine hydroxylase in a direct binding assay [25]. Further- more, the Hill coefficient for H 4 biopterin binding to hPAH revealed a dependence (both for the isolated tetrameric wt-hPAH and the tetrameric fusion protein MBP-(D 4 K) ek - hPAH) on the induction time with IPTG in E. coli,i.e.a short induction period (2 h at 28 °C) gave a slightly higher Hill coefficient than 24 h induction (data not shown). This finding may be related to the differences observed in our steady-state kinetic analyses of the two enzyme forms [10]. Thus, the tetrameric wt-hPAH isolated after 2 h and 24 h of induction with IPTG in E. coli, revealed differences (24 h vs. 2 h) in both the affinity for the cofactor H 4 biopterin (decreased) and L-Phe (increased), as well as for the catalytic efficiency (increased) [10]. The main physico-chemical difference between these two enzyme preparations was the extent of time-dependent nonenzymatic deamidation (dur- ing expression in E. coli) of specific labile Asn residues [10]. Complementary mutagenesis (AsnfiAsp) analyses have demonstrated that the rate of phosphorylation is indeed dependent on the extent of deamidation of a very labile Asn residue (Asn32) in the N-terminal autoregulatory sequence of wt-hPAH [34]. Dopamine has a dual mechanism for its inhibition of PAH. First, the bidentate coordination of its catechol hydroxyl groups to the active site iron [17] results in a complex with strong inhibition of the catalytic activity [32]. Secondly, on binding at the active site, the dopamine main- chain interacts with the autoregulatory sequence at Ser23 (by hydrogen bonding) and Ile25 (Fig. 4), which results in an inhibition of Ser16 phosphorylation similar to that of H 4 biopterin (Fig. 3B) with a half-maximum inhibition at a concentration < 0.1 l M . This value compares well with the concentration determined for the half-maximum binding (0.25 l M ) of noradrenaline to rPAH [32]. A similar type of interaction between dopamine and the regulatory domain of the structurally and functionally closely related enzyme tyrosine hydroxylase has been reported in experiments using limited proteolysis as a structural probe [35]. In this case, the affinity of dopamine binding was even higher than in PAH, with a K d ¼ 1.3 ± 0.6 n M , which is considered to be of physiological relevance (reviewed in [36]). The interdependent binding of biopterin cofactor and amino acid substrate Internal protein dynamics are intimately connected to the catalytic activity of PAH, and our recent structural studies on hPAH have revealed some key features of its hysteretic properties. Thus, the structures of the binary and ternary complexes of hPAH have provided a detailed picture of the protein contacts involved in the binding of biopterin cofactor and amino acid substrate at distinctly different positions in the active site crevice structure [20,21]. More- over, the cystal structures have revealed that the binding of both biopterin cofactor and substrate is accompanied by local (active site) conformational changes, most pronounced for the binding of L-Phe, which also triggers global conformational changes (hysteresis) in the protomer as determined by SPR analyses (Fig. 2 [26,27]). However, as there is still no crystal structure available for the full-length form of the ligand-free enzyme and the binary substrate complex, it is not known how the observed conformational changes at the active site [21] are propagated to the rest of the full-length protomer in the oligomeric forms. The structural changes observed at the active site of the catalytic domain structure [21] explain why the binding of L-Phe reduces the affinity of pterin cofactor binding (Fig. 4C) and vice versa (Fig. 2). Thus, the large conformational change observed at the active site on substrate binding changes the position and orientation (relative to the catalytic iron) and the hydrogen- bonding network of the bound H 4 biopterin. Moreover, the substrate induced repositioning of the cofactor also accounts for the release of its interaction with the autoregulatory sequence (Fig. 4C) and the related inhibition of Ser16 phosphorylation (Fig. 3). A similar effect of substrate binding was observed for the dopamine inhibition of phosphorylation (Fig. 3) and of the limited proteolysis by trypsin (Fig. 1), which are both completely reversed by L-Phe binding. The interdependent binding properties of the pterin cofactor and L-Phe have also been observed in kinetic studies on mutant forms of hPAH (point mutations of active site residues), which often have a profound influence on biopterin cofactor and/or substrate binding affinity [18, 37–40]. If the affinity for the cofactor is reduced, that of the substrate is often observed to be increased, and vice versa [18,40]. It should be noted that enzyme kinetic, three- dimensional structural and biophysical (MCD and EXAFS) studies all support an ordered reaction mechanism wherein both cofactor and substrate must be bound before reaction with dioxygen can occur to generate the active intermediate for the coupled hydroxylation of the cosubstrates [21,41]. However, the sequence of binding of cofactor and substrate is still a matter of discussion and may be different comparing wild-type and mutant forms of the enzyme. Acknowledgements The study was supported by grants from the Research Council of Norway (NFR), from The Novo Nordisk Foundation, The Nansen Fund, The Blix Family Fund for Advancement of Medical Research and the Norwegian Council on Cardiovascular Diseases. We greatly appreciate the expert technical assistance of Ali Sepulveda Mun ˜ oz in expression and purification of the recombinant enzymes, and the staff of the Swiss-Norwegian Beamlines in Grenoble (France). References 1. Kaufman, S. (1971) The phenylalanine hydroxylating system from mammalian liver. Adv. Enzymol. Relat. Areas Mol. Biol. 35, 245– 319. 988 T. Solstad et al.(Eur. J. Biochem. 270) Ó FEBS 2003 2. Richardson, S.C. & Fisher, M.J. (1993) Characterization of phenylalanine hydroxylase from rat kidney. Int. J. Biochem. 25, 581–588. 3. Lichter-Konecki, U., Hipke, C.M. & Konecki, D.S. (1999) Human phenylalanine hydroxylase gene expression in kidney and other nonhepatic tissues. Mol. Genet. Metab. 67, 308–316. 4. Schallreuter,K.U.,Lemke,K.R.,Pittelkow,M.R.,Wood,J.M., Korner, C. & Malik, R. (1995) Catecholamines in human kerati- nocyte differentiation. J. Invest. Dermatol. 104, 953–957. 5. Scriver, C.R., Waters, P.J., Sarkissian, C., Ryan, S., Prevost, L., Cote, D., Novak, J., Teebi, S. & Nowacki, P.M. (2000) PAHdb: a locus-specific knowledgebase. Hum. Mutat. 15, 99–104. 6. Shiman,R.,Mortimore,G.E.,Schworer,C.M.&Gray,D.W. (1982) Regulation of phenylalanine hydroxylase activity by phenylalanine in vivo, in vitro, and in perfused rat liver. J. Biol. Chem. 257, 11213–11216. 7. Shiman, R. & Gray, D.W. (1980) Substrate activation of phenyl- alanine hydroxylase. A kinetic characterization. J. Biol. Chem. 255, 4793–4800. 8. Shiman, R., Jones, S.H. & Gray, D.W. (1990) Mechanism of phenylalanine regulation of phenylalanine hydroxylase. J. Biol. Chem. 265, 11633–11642. 9. Kaufman, S. (1987) Phenylalanine 4-monooxygenase from rat liver. Methods Enzymol. 142, 3–17. 10. Solstad, T. & Flatmark, T. (2000) Microheterogeneity of recombinant human phenylalanine hydroxylase as a result of nonenzymatic deamidations of labile amide containing amino acids. Effects on catalytic and stability properties. Eur. J. Biochem. 267, 6302–6310. 11. Xia, T., Gray, D.W. & Shiman, R. (1994) Regulation of rat liver phenylalanine hydroxylase. III. Control of catalysis by (6R)- tetrahydrobiopterin and phenylalanine. J. Biol. Chem. 269, 24657– 24665. 12. Døskeland, A.P., Døskeland, S.O., Øgreid, D. & Flatmark, T. (1984) The effect of ligands of phenylalanine 4-monooxygenase on the cAMP-dependent phosphorylation of the enzyme. J. Biol. Chem. 259, 11242–11248. 13. Døskeland, A.P., Martı ´ nez, A., Knappskog, P.M. & Flatmark, T. (1996) Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease. Biochem. J. 313, 409–414. 14. Shiman, R., Xia, T., Hill, M.A. & Gray, D.W. (1994) Regulation of rat liver phenylalanine hydroxylase. II. Substrate binding and the role of activation in the control of enzymatic activity. J. Biol. Chem. 269, 24647–24656. 15. Shiman, R., Gray, D.W. & Hill, M.A. (1994) Regulation of rat liver phenylalanine hydroxylase. I. Kinetic properties of the enzyme’s iron and enzyme reduction site. J. Biol. Chem. 269, 24637–24646. 16. Erlandsen, H., Fusetti, F., Martı ´ nez, A., Hough, E., Flatmark, T. & Stevens, R.C. (1997) Crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria. Nat. Struct. Biol. 4, 995–1000. 17. Erlandsen, H., Flatmark, T., Stevens, R.C. & Hough, E. (1998) Crystallographic analysis of the human phenylalanine hydroxylase catalytic domain with bound catechol inhibitors at 2.0 A ˚ resolu- tion. Biochemistry 37, 15638–15646. 18. Erlandsen, H., Bjørgo, E., Flatmark, T. & Stevens, R.C. (2000) Crystal structure and site-specific mutagenesis of pterin- bound human phenylalanine hydroxylase. Biochemistry 39, 2208– 2217. 19. Kobe, B., Jennings, I.G., House, C.M., Michell, B.J., Goodwill, K.E.,Santarsiero,B.D.,Stevens,R.C.,Cotton,R.G.&Kemp, B.E. (1999) Structural basis of autoregulation of phenylalanine hydroxylase. Nat. Struct. Biol. 6, 442–448. 20. Andersen, O.A., Flatmark, T. & Hough, E. (2001) High resolution crystal structures of the catalytic domain of human phenylalanine hydroxylase in its catalytically active Fe(II) form and binary complex with tetrahydrobiopterin. J. Mol. Biol. 314, 279–291. 21. Andersen, O.A., Flatmark, T. & Hough, E. (2002) Crystal struc- ture of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2- thienyl)- L -alanine, and its implications for the mechanisms of catalysis and substrate activation. J. Mol. Biol. 320, 1095–1108. 22. Teigen, K., Frøystein, N.A. & Martı ´ nez, A. (1999) The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism. J. Mol. Biol. 294, 807–823. 23. Martı ´ nez, A., Knappskog, P.M., Olafsdottir, S., Døskeland, A.P., Eiken, H.G., Svebak, R.M., Bozzini, M., Apold, J. & Flatmark, T. (1995) Expression of recombinant human phenylalanine hydro- xylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme. Biochem. J. 306, 589–597. 24. Roskoski, R. Jr (1983) Assays of protein kinase. Methods Enzy- mol. 99,3–6. 25. Flatmark, T., Alma ˚ s, B., Knappskog, P.M., Berge, S.V., Svebak, R.M., Chehin, R., Muga, A. & Martı ´ nez, A. (1999) Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection. Eur. J. Biochem. 262, 840–849. 26. Flatmark, T., Stokka, A.J. & Berge, S.V. (2001) Use of surface plasmon resonance for real-time measurements of the global conformational transition in human phenylalanine hydroxylase in response to substrate binding and catalytic activation. Anal. Bio- chem. 294, 95–101. 27. Stokka, A.J. & Flatmark, T. (2003) Substrate induced con- formational transition in human phenylalanine hydroxylase as studied by surface plasmon resonance analyses. The effect of terminal deletions, substrate analogues and phosphorylation. Biochem. J. 369, 509–518. 28. Jo ¨ nsson, U., Fa ¨ gerstam, L., Ivarsson, B., Johnsson, B., Karlsson, R., Lundh, K., Lo ¨ fas,S.,Persson,B.,Roos,H.,Ro ¨ nnberg, I., Sjo ¨ lander,S.,Stenberg,E.,Sta ˚ hlberg, R., Urbaniczky, C., O ¨ stlin, H. & Malmqvist, M. (1991) Real–time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Bio Techniques 11, 620–627. 29. Phillips, R.S., Iwaki, M. & Kaufman, S. (1983) Ligand effects on the limited proteolysis of phenylalanine hydroxylase: evidence for multiple conformational states. Biochem. Biophys. Res. Commun. 110, 919–925. 30. Iwaki, M., Phillips, R.S. & Kaufman, S. (1986) Proteolytic modification of the amino-terminal and carboxyl-terminal regions of rat hepatic phenylalanine hydroxylase. J. Biol. Chem. 261, 2051–2056. 31. Flatmark, T. & Stevens, R.C. (1999) Structural insight into the aromatic amino acid hydroxylases and their disease related mutant forms. Chem. Rev. 99, 2137–2360. 32. Martı ´ nez, A., Haavik, J. & Flatmark, T. (1990) Cooperative homotropic interaction of 1-noradrenaline with the catalytic site of phenylalanine 4-monooxygenase. Eur. J. Biochem. 193, 211–219. 33. Miranda, F.F., Teigen, K., Tho ´ ro ´ lfsson, M., Svebak, R.M., Knappskog, P.M., Flatmark, T. & Martı ´ nez, A. (2003) Phos- phorylation and mutations of Ser16 in human phenylalanine hydroxylase. Kinetic and structural effects. J. Biol. Chem. 277, 40937–40943. 34. Carvalho, R.N., Solstad, T., Bjørgo, E., Barroso, J.F. & Flatmark, T. (2003) Deamidations in recombinant human phenylalanine hydroxylase. Identification of labile asparagine residues and functional characterization of AsnfiAsp mutant forms. J. Biol. Chem. [epub ahead of print on Jan 28]. Ó FEBS 2003 Regulatory properties of phenylalanine hydroxylase (Eur. J. Biochem. 270) 989 35. McCulloch, R.I. & Fitzpatrick, P.F. (1999) Limited proteolysis of tyrosine hydroxylase identifies residues 33–50 as conformationally sensitive to phosphorylation state and dopamine binding. Arch. Biochem. Biophys. 367, 143–145. 36. Flatmark, T. (2000) Catecholamine biosynthesis and physiological regulation in neuroendocrine cells. Acta Physiol. Scand. 168, 1–17. 37. Knappskog, P.M., Eiken, H.G., Martı ´ nez, A., Bruland, O., Apold, J. & Flatmark, T. (1996) PKU mutation (D143G) asso- ciated with an apparent high residual enzyme activity: expression of a kinetic variant form of phenylalanine hydroxylase in three different systems. Hum. Mutat. 8, 236–246. 38. Bjørgo, E., Knappskog, P.M., Martı ´ nez,A.,Stevens,R.C.& Flatmark, T. (1998) Partial characterization and three-dimen- sional-structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenyl- ketonuria. Eur. J. Biochem. 257, 1–10. 39. Leandro,P.,Rivera,I.,Lechner,M.C.,deAlmeida,I.T.&Konecki, D. (2000) The V388M mutation results in a kinetic variant form of phenylalanine hydroxylase. Mol. Genet. Metab. 69, 204–212. 40. Jennings, I.G., Cotton, R.G. & Kobe, B. (2000) Functional ana- lysis, using in vitro mutagenesis,ofaminoacidslocatedinthe phenylalanine hydroxylase active site. Arch. Biochem. Biophys. 384, 238–244. 41. Solomon,E.I.,Brunold,T.C.,Davis,M.I.,Kemsley,J.N.,Lee, S K., Lehnert, N., Neese, F., Skulan, A.J., Yang, Y S. & Zhou, J. (2000) Geometric and electronic structure/function correlations in non-heme iron enzymes. Chem. Rev. 100, 235–350. 42. Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crys- tallogr. 24, 946–950. 990 T. Solstad et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . Studies on the regulatory properties of the pterin cofactor and dopamine bound at the active site of human phenylalanine hydroxylase Therese Solstad 1 ,. at the active site on substrate binding changes the position and orientation (relative to the catalytic iron) and the hydrogen- bonding network of the bound