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Supporting Text
Site-Directed Mutagenesis and Purification. The mutations F39L, I65T, R68S, H170D, E178G, V190A, R261Q, A300S, L308F, A313T, A373T, V388M, E390G, P407S, and Y414C were introduced into the pMAL-c2 plasmid containing the full-length WT PAH (wt-PAH) sequence by using the QuikChange site-directed mutagenesis kit (Stratagene). The primers used are shown in Table 3. The authenticity of the mutagenesis was verified by DNA sequencing. The mutations I65T, R68S, R261Q, V388M, and Y414C in full-length phenylalanine hydroxylase (PAH) have already been reported (1-4). In addition, four of the mutations, i.e., H170D, R261Q, A313T, and Y414C, were introduced into the double-truncated mutant form (D N1–102/D C428–452; dt-PAH) of human PAH (5). Growth of Escherichia coli transformed with the pMAL vectors for expression of full-length wt-PAH and mutant enzymes and purification of the tetrameric forms were performed as described (5, 6). The dimeric dt-PAH enzymes were also purified in the same way. Protein concentration was measured spectrophotometrically by using e 280 nm (1 mg· ml-1) = 1.63 cm-1 for the myelin basic protein (MBP)-fusion protein and e 280 nm (1 mg· ml-1) = 1.0 cm-1 for isolated PAH (6).
Measurement of Mutant and wt-PAH Activity. Measurement of mutant and wt-PAH activity by HPLC and fluorescence detection of L-Tyr for nonactivated (non-L-Phe preincubated) and activated (L-Phe preincubated, 1 mM L-Phe) PAH enzymes was measured as described (5). The degree of activation (fold) refers to the ratio between the specific activities of the L-Phe-preincubated and nonpreincubated samples. The kinetic parameters were calculated by nonlinear regression analysis of the experimental data by using the Hill equation.
To determine the coupling efficiency of the reaction, i.e., ratio L-Tyr formation/(6R)-<cm;1>l<cm;0>-erythro-5,6,7,8-tetrahydrobiopterin (BH4) oxidation, the enzyme (0.1-0.2 mg/ml) was incubated at room temperature for 4 min in 200 mM Na-Hepes, pH 7.0/0.04 mg/ml catalase/200 m M NADH/0.4 units dihydropteridine reductase (from sheep liver, Sigma)/1 mM L-Phe in a final volume of 1 ml. Then ferrous ammonium-sulfate was added (100 m M), and after 1 min, the reaction was initiated by adding 6R-BH4 (75 m M; from Schirck’s Laboratories, Jona, Switzerland). D A340 was measured in the first minute, and 50 m l of the reaction was taken to measure L-Tyr formation by fluorimetric detection (see above). The rate of NADH oxidation (BH4 oxidation) was calculated by using e 340 nm = 6220 M-1· cm-1. Under these conditions, BH4 consumption and L-Tyr formation were linear with respect to the reaction time and amount of protein used.
Isothermal Titration Calorimetry (ITC). The experiments were performed in a VP-ITC titration calorimeter (MicroCal, Amherst, MA) at pH 7.0 with the glucose oxidase system (7) and 10-50 m M PAH subunit with » 0.5 mol ferrous ammonium sulfate/subunit, as described (3).
Circular Dichroism (CD). Thermal denaturation was monitored by following the changes in ellipticity at 222 nm at a scan rate of 0.7 K/min in the temperature range 30-70°C by using a Jasco (Easton, MD) J-810 spectropolarimeter equipped with a Jasco Peltier 423S element. Measurements were performed at pH 7.0 with purified tetrameric enzyme (10 m M subunit) and equimolar amounts of ferrous ammonium-sulfate per enzyme subunit.
In Vitro Transcription-Translation Synthesis. PAH protein was synthesized in vitro by using the TnT-T7 transcription-translation system from Promega in the absence or presence of 500 m M BH4, as described (3, 4).
Crystallization and Data Collection. Purified protein at a concentration of » 10 mg/ml of the four mutants (H170D, R261Q, A313T, and Y414C) of dt-PAH was used for crystallization trials. Only the A313T-dt-PAH mutant crystallized under similar conditions as for the double-truncated wt-PAH (8, 9). The crystallization conditions were 5–10% ethylene glycol/20-40 mM Pipes, pH 6.8/8–15% polyethylene glycol 2000. Two data sets, one without 7,8-BH2 (denoted A313T-dt-PAH) and one with 7,8-BH2 cocrystallized (plus additionally soaked into the crystals at a concentration of » 6 mM, denoted A313T-dt-PAH•7,8-BH2), were collected by using cryo-cooled crystals frozen in liquid nitrogen on an in-house FRD generator (Rigaku/MSC, Tokyo) (wavelength = 1.5418 Å) with an R-axis IV++ image plate detector, by using Osmic mirrors and an X-stream cryostat set to 100 K. The images were collected at a 1° oscillation angle and a crystal-to-detector distance of 170 mm.
Data Processing, Model Building, and Refinement. Data were processed with HKL2000 (10) to 2.1 and 2.2 Å, for the A313T-dt-PAH and A313T-dt-PAH•7,8-BH2, respectively. The structure of the native double-truncated form of human PAH [Protein Data Bank (PDB) code 1PAH] (8) and the structure of the double-truncated form of human PAH with 7,8-BH2 bound (PDB code 1LRM) (11) were used as models to calculate difference electron density maps (Fobs-Fcalc) for the A313T-dt-PAH and the A313T-dt-PAH•7,8-BH2 structures, respectively. Electron density for a threonine residue was clear in the initial maps in both structures, and the entire 7,8-BH2 ligand was readily visible in electron density. Map calculations, energy minimization, and refinement were performed by using the program CNS (Version 1.1) (12). The program O (Version 7) (13) was used to manually fit the differences from the native structures into the experimental electron density, using CNS composite s A-weighted omit maps as well as regular 2Fobs-Fcalc and Fobs-Fcalc maps. The final model statistics are shown in Table 6.
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