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: Km, 6-methyltetrahydropterin = 61 microM and L-Phe = 170 microM; Vmax = 9 s-1 (compared to 45 microM, 180 microM, and 13 s-1 for the rat hepatic enzyme). L-Phe and lysolecithin treatment induce the rPAHT-->rPAHR (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 Eact 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 Eact 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 ST = 3/2 spin system.
Kappock
T J
TJ
Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, USA.
Harkins
P C
PC
Friedenberg
S
S
Caradonna
J P
JP
D000494:Allosteric Regulation; D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D046911:Macromolecular Substances; D008970:Molecular Weight; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics
D008244:Lysophosphatidylcholines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010651:Phenylalanine Hydroxylase