The major objective of this research is to elucidate the chemical and physical properties of the mononuclear non-heme iron dependent metalloenzyme, phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase, E.C. 18.104.22.168). PAH is a mixed-function oxidase whose active site ferrous iron center catalyzes the hydroxylation of L-phenylalanine (L-phe) to L-tyrosine (L-tyr) in the presence of dioxygen and the reduced cofactor, tetrahydropterin. Genetic defects in the PAH gene that induce a significant decrease in PAH activity lead to the autosomal recessive human genetic disorder, classic phenylketonuria (PKU). This disorder is characterized by an increase in serum L-phe concentrations and the abnormal accumulation of phenylalanine-based metabolic products that are thought to cause defective myelination of the central nervous system, leading to postnatal brain damage and severe mental retardation. PKU is the most common inborn error in amino acid metabolism that is of clinical importance; approximately 1 in 50 individuals carry the disease trait with an average incidence of about 1 in 10,000 for Caucasians. Early detection and strict dietary management have significantly reduced the neurological defects and mental retardation characteristics of untreated PKU. Among the specific aims of this project is to identify and interpret, in terms of their structural and chemical implications, changes in the active site iron environment as a function of the requisite allosteric activation process, and the binding of substrate(s) and cofactor. Advanced methodologies, including MCD, XAS, M?ssbauer, ESEEM, and ENDOR spectroscopies, will be utilized to probe the active site structures of multiple physiologically relevant enzyme states, all of which can be generated in homogeneous forms. A comparison of the active site characteristics of native, homotetrameric PAH with dimeric/monomeric forms of the enzyme will be performed; truncated dimeric forms of PAH support promiscuous oxidation chemistry in terms of which substrates can be oxidized versus wildtype full length PAH, which only efficiently accepts L-phe as substrate. Additional objectives are to compare wildtype PAH with selected PKU inducing missense mutations as a further probe of PAH mechanism and to initiate studies designed to defining the chemical basis for hyperphenylalaninemic disorders. Finally, the use of double mixing stopped-flow UV/vis and florescence experiments will be utilized to probe the compulsory reduction of the ferric active site prior to catalytic oxidation of substrate. Kinetic and mechanistic experiments will be performed to identify the factors that control and regulate iron reduction.
|Kemsley, Jyllian N; Wasinger, Erik C; Datta, Supratim et al. (2003) Spectroscopic and kinetic studies of PKU-inducing mutants of phenylalanine hydroxylase: Arg158Gln and Glu280Lys. J Am Chem Soc 125:5677-86|
|Foster, Trina L; Caradonna, John P (2003) Fe2+-catalyzed heterolytic RO-OH bond cleavage and substrate oxidation: a functional synthetic non-heme iron monooxygenase system. J Am Chem Soc 125:3678-9|
|Wasinger, Erik C; Mitic, Natasa; Hedman, Britt et al. (2002) X-ray absorption spectroscopic investigation of the resting ferrous and cosubstrate-bound active sites of phenylalanine hydroxylase. Biochemistry 41:6211-7|