Reduction and oxidation of the active site iron in tyrosine hydroxylase: kinetics and specificity.
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.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
Patrick A
PA
Frantom
Javier
J
Seravalli
Stephen W
SW
Ragsdale
Paul F
PF
Fitzpatrick
D000693:Anaerobiosis; D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction; D011622:Pterins / Q000737:chemistry; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics
D011622:Pterins; D001708:Biopterins; C017523:6-methyltetrahydropterin; D007501:Iron; D014446:Tyrosine 3-Monooxygenase; C003402:sapropterin; D001205:Ascorbic Acid