Reduction and oxidation of the active site iron in tyrosine hydroxylase: Kinetics and specificity

Patrick A. Frantom, Javier Seravalli, Stephen W. Ragsdale, Paul F. Fitzpatrick

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze-quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 ± 0.1 mM-1 s-1. 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 ± 0.1 mM -1 s-1 and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated Km for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH4 does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM-1 s-1. S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.

Original languageEnglish (US)
Pages (from-to)2372-2379
Number of pages8
JournalBiochemistry
Volume45
Issue number7
DOIs
StatePublished - Feb 21 2006

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Tyrosine 3-Monooxygenase
Catalytic Domain
Iron
Oxidation
Kinetics
Reducing Agents
Enzymes
Pterins
Rate constants
Paramagnetic resonance
Hydroxylation
Dihydroxyphenylalanine
Phosphorylation
Molecular oxygen
Kinetic parameters
Oxidation-Reduction
Glutathione
Tyrosine
Conformations
Spectrum Analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reduction and oxidation of the active site iron in tyrosine hydroxylase : Kinetics and specificity. / Frantom, Patrick A.; Seravalli, Javier; Ragsdale, Stephen W.; Fitzpatrick, Paul F.

In: Biochemistry, Vol. 45, No. 7, 21.02.2006, p. 2372-2379.

Research output: Contribution to journalArticle

Frantom, Patrick A. ; Seravalli, Javier ; Ragsdale, Stephen W. ; Fitzpatrick, Paul F. / Reduction and oxidation of the active site iron in tyrosine hydroxylase : Kinetics and specificity. In: Biochemistry. 2006 ; Vol. 45, No. 7. pp. 2372-2379.
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N2 - Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze-quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 ± 0.1 mM-1 s-1. 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 ± 0.1 mM -1 s-1 and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated Km for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH4 does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM-1 s-1. S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.

AB - Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze-quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 ± 0.1 mM-1 s-1. 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 ± 0.1 mM -1 s-1 and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated Km for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH4 does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM-1 s-1. S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.

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