Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase make up the family of non-heme iron pterin-dependent aromatic amino acid hydroxylases. Each enzyme catalyzes the hydroxylation of the aromatic side chain of its substrate in a reaction critical for proper functioning of the central nervous system: phenylalanine metabolism, catecholamine biosynthesis, and serotonin biosynthesis. In addition to their physiological importance, these enzymes are unique in that they are the only nonheme iron enzymes whose physiological reaction is the hydroxylation of unactivated aromatic systems. The present proposal addresses several outstanding questions regarding the catalytic and regulatory mechanisms of these enzymes. The common catalytic mechanism will be studied using a combination of rapid reaction methods and spectroscopy, with the goal of characterizing individual iron species in catalysis. The role of protein dynamics in catalysis by tyrosine and phenylalanine hydroxylase will be studied by determining the effects of substrates on the kinetics of peptide bond exchange with solvent. The regulation of phenylalanine and tyrosine hydroxylase is critical to their proper function. The kinetics of peptide bond exchange will be used to evaluate a literature model for allosteric regulation of phenylalanine hydroxylase, while fluorescence methods will be used to evaluate a structural model for the regulation of tyrosine hydroxylase by phosphorylation. The active site iron atom in these enzymes is bound by a 2-Histidine-1-carboxylate facial triad. These two histidines in phenylalanine hydroxylase will be replaced with analogs and the effects on individual kinetic steps determined, using a combination of steady state kinetics, product partitioning, and kinetic isotope effects. The aromatic amino acid hydroxylases play critical roles in the proper functioning of the central nervous system. Loss of phenylalanine hydroxylase results in phenylketonuria;tyrosine hydroxylase is required to make the neurotransmitters dopamine, norepinephrine, and epinephrine;and tryptophan hydroxylase is required to synthesize the neurotransmitter serotonin.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047291-20
Application #
7994171
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Anderson, Vernon
Project Start
1995-07-01
Project End
2013-07-31
Budget Start
2010-12-01
Budget End
2013-07-31
Support Year
20
Fiscal Year
2011
Total Cost
$325,756
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Zhang, Shengnan; Fitzpatrick, Paul F (2016) Identification of the Allosteric Site for Phenylalanine in Rat Phenylalanine Hydroxylase. J Biol Chem 291:7418-25
Fitzpatrick, Paul F (2015) Structural insights into the regulation of aromatic amino acid hydroxylation. Curr Opin Struct Biol 35:1-6
Zhang, Shengnan; Huang, Tao; Ilangovan, Udayar et al. (2014) The solution structure of the regulatory domain of tyrosine hydroxylase. J Mol Biol 426:1483-97
Roberts, Kenneth M; Pavon, Jorge Alex; Fitzpatrick, Paul F (2013) Kinetic mechanism of phenylalanine hydroxylase: intrinsic binding and rate constants from single-turnover experiments. Biochemistry 52:1062-73
Krzyaniak, Matthew D; Eser, Bekir E; Ellis, Holly R et al. (2013) Pulsed EPR study of amino acid and tetrahydropterin binding in a tyrosine hydroxylase nitric oxide complex: evidence for substrate rearrangements in the formation of the oxygen-reactive complex. Biochemistry 52:8430-41
Roberts, Kenneth M; Fitzpatrick, Paul F (2013) Mechanisms of tryptophan and tyrosine hydroxylase. IUBMB Life 65:350-7
Daubner, S Colette; Avila, Audrey; Bailey, Johnathan O et al. (2013) Mutagenesis of a specificity-determining residue in tyrosine hydroxylase establishes that the enzyme is a robust phenylalanine hydroxylase but a fragile tyrosine hydroxylase. Biochemistry 52:1446-55
Fitzpatrick, Paul F (2012) Allosteric regulation of phenylalanine hydroxylase. Arch Biochem Biophys 519:194-201
Panay, Aram Joel; Lee, Michael; Krebs, Carsten et al. (2011) Evidence for a high-spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase. Biochemistry 50:1928-33
Li, Jun; Ilangovan, Udayar; Daubner, S Colette et al. (2011) Direct evidence for a phenylalanine site in the regulatory domain of phenylalanine hydroxylase. Arch Biochem Biophys 505:250-5

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