Structural Studies of the Catalytic and Regulatory Mechanisms of Phenylalanine Hydroxylase
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The catalytic and regulatory mechanisms of phenylalanine hydroxylase were investigated by structural studies of in this research. Phenylalanine hydroxylase (PheH) hydroxylates phenylalanine to produce tyrosine using tetrahydrobiopterin (BH4) and oxygen. The three ligands to the iron, His285, His290, and Glu330, were mutated to glutamine, glutamate, and histidine. All the mutants had low but measurable activity. Mutation of Glu330 had the greatest effect on activity and mutation of His290 the least. All the mutations resulted in an excess of tetrahydropterin oxidized relative to tyrosine formation, with mutation of His285 having the greatest effect on the coupling of the two partial reactions. All the mutants greatly decreased the affinity for iron, with mutation of Glu330 the most deleterious. The results complement previous results with tyrosine hydroxylase in establishing the plasticity of the individual iron ligands in this enzyme family. Hydrogen/deuterium exchange and mass spectrometry showed that peptides lying in the interface between the regulatory and catalytic domains display large increases of deuterium incorporation in the presence of phenylalanine. However, the effects of phenylalanine on a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site of phenylalanine. These results support the autoinhibitory function of the N-terminus of PheH. No peptides show a changed deuterium incorporation pattern in the presence of BH4, suggesting that BH4 binding does not change the structure significantly from the resting form. In phosphorylated PheH, three peptides show a deuterium incorporation pattern similar to that of unphosphorylated PheH plus phenylalanine, while the other peptides sensitive to phenylalanine binding in unphosphorylated PheH show the same pattern as that of unphosphorylated PheH without phenylalanine. Therefore, the conformational changes induced by phosphorylation are similar to but not identical to those associated with phenylalanine activation. The isolated regulatory domain (PheH1-117) was expressed and purified using a QSepharose column followed by a gel filtration column. Analytical gel filtration shows that PheH1-117 exists as a dimer in solution. In the presence of phenylalanine, the retention time of PheH1-117 is significantly changed. The 1H-15N NMR spectra of PheH1- 117 show that the cross-peaks of several residues are altered in the presence of phenylalanine. These results support the existence of a regulatory binding site for phenylalanine in the regulatory domain of PheH.
Li, Jun (2010). Structural Studies of the Catalytic and Regulatory Mechanisms of Phenylalanine Hydroxylase. Doctoral dissertation, Texas A&M University. Available electronically from