| The aromatic amino acid monooxygenase family consists of phenylalanine, tyrosine, and tryptophan hydroxylases (PAH, TYH, TrpH). These non-heme, iron-dependent enzymes require molecular oxygen and tetrahydrobiopterin for catalytic activity and play essential roles in the biosynthetic pathways of neurotransmitters. Dysfunction of PAH can cause phenylketonuria (PKU). Specific PAH mutants were investigated in order to further the mechanistic understanding of PAH and its dysfunction, as well as to exploit unique properties to trap the currently unobserved ferryl-oxo hydroxylating intermediate. The TYH project was launched to gain chemical insight into the neurological and psychological disorders associated with dopameric irregularities caused by TYH dysfunction.; Truncated PAH (Delta1-117PAH), lacking the regulatory domain, eliminated restricted access to the iron active site and provided insight into the function of that domain. Mutation of Ser349 to Ala disrupted a key hydrogen bond to the His285 iron ligand that locks the substrate-binding site to the iron site. The PAH S349A mutant was created to capitalize on the resultant slow product formation in order to observe the iron-oxo intermediate by stopped-flow spectroscopy. The Delta1-117PAH S349A mutant, which exploits both the decoupled hydroxylation reaction and the increased active site access, was successfully used to trap a ferryl intermediate through rapid-freeze-quench Mossbauer spectroscopy.; Mutants Delta1-117PAH, PAH S349A, and Delta1-117PAH S349A were characterized and compared to wtPAH, especially with regards to the active site geometry, kinetics, mechanism, and structure. The results revealed key mechanistic properties that resembled wtPAH: an NIH shift, formation of the C4a -hydroxypterin species, pterin utilization tightly coupled to product formation, as well as similar MCD spectra that verified specific geometric states of the enzyme. The catecholate-Fe+2 LMCT band produced by Delta1-117PAH and Delta1-117PAH S349A was hypsochromically shifted compared to wtPAH and indicated changes in the ligand environment of the iron active site of the two truncated mutants. Both S349A-containing mutants exhibited only 10% the activity of wtPAH and displayed Km, L-phe values that were an order of magnitude larger than for wtPAH. The results discussed in this thesis provide insight into the reaction mechanism of phenylalanine hydroxylase and chemical basis of PAH dysfunction. |
