Reduction and Oxidation of the Active Site Iron in Tyrosine Hydroxylase:  Kinetics and Specificity

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 K m for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH 4 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.Tyrosine hydroxylase (TyrH) 1 is a pterin-dependent monooxygenase that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine (DOPA) using a non-heme iron atom in the active site to activate molecular oxygen (1). Formation of DOPA is the first and rate-limiting step in the biosynthesis of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Because of the important physiological role played by TyrH, its activity is regulated at the posttranslational level by phosphorylation of serine residues and by feedback inhibition by catecholamines. The redox state of the iron plays a central role in this regulation, as shown in Scheme 1 (2,3). For catalysis, the active site iron must be ferrous (4); during catalysis it is oxidized to higher levels but returns to the ferrous form by the end of the catalytic cycle (5). However, in a side reaction independent of catalysis, oxygen can oxidize the ferrous iron to the inactive ferric form (2); the iron must then be reduced back to the ferrous † This work was funded in part by NIH Grants R01 GM47291 (to P.F.F.), T32 GM08523 (to P.A.F.), and R01 GM39451 (to S.W.R.).

Selectivity and Affinity Determinants for Ligand Binding to the Aromatic Amino Acid Hydroxylases

“…Uncoupling of the reaction generates H 2 O 2 , which may cause oxidative stress to the cell, and it is imperative to take uncoupling into account when considering cofactor analogs for use in cofactor replacement therapy. A direct pathogenetic role of TH in Parkinson's disease has been suggested [15], as the enzyme is a source of reactive oxygen species in vitro [10,99]. The substrate binding mode for L-Phe in PAH and for LTrp in TPH1 has also been solved by NMR and docking [80,83].…”

“…Each enzyme catalyzes the hydroxylation of its respective amino acid substrate, using molecular oxygen and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) as additional substrates. In order to perform catalysis, these enzymes are dependent on a nonheme iron [5][6][7] and the pterin co-substrate, often referred to as cofactor, which also reduces the active site iron from the inactive ferric state to the active ferrous form [8][9][10]. Phenylalanine (L-Phe) is hydroxylated in the para position to tyrosine (L-Tyr) by PAH; L-Tyr is hydroxylated in the meta position by TH to 3,4-dihydroxyphenylalanine (DOPA); and TPH1 and TPH2 hydroxylate tryptophan (LTrp) to 5-hydroxytryptophan (5-OH-Trp).…”

Dynamics of tyrosine hydroxylase mediated regulation of dopamine synthesis

“…Frantom et al (2006) Qualitative/Quantitative Discusses the effects of the reduction and oxidation of the iron molecule of TH. Includes oxidation of the cofactor and its regeneration from its metabolites.…”

“…While it is sensitive to changes even in its approach to steady state, it responds more quickly after steady state has been achieved, a condition corresponding more closely to in-vivo physiology. 4.5 Comparison of this model with existing models of TH activity Several attempts have been made in the past to model Tyrosine Hydroxylase's modulatory effects on catecholamine synthesis Justice et al, 1988;Fitzpatrick, 1991;Ramsey and Fitzpatrick, 1998;Frantom et al, 2006) but none up to now have included phosphorylation, oxidative regulation, alpha synuclein and feedback inhibition. Table 1 summarizes the similarities and differences between this kinetic model and the previously published regulatory models for tyrosine hydroxylase activity.…”

“…[E2up] = 0 nM k f = 210 (mM −1 ) × (s −1 ) (Frantom et al, 2006) Modeled as an irreversible forward reaction with a single rate constant. Y is a hypothetical entity which could be superoxide.…”