Reduction and oxidation of the active site iron in tyrosine hydroxylase: Kinetics and specificity
Reduction and oxidation of the active site iron in tyrosine hydroxylase: Kinetics and specificity
复制标题
DOI：
10.1021/bi052283j
复制
发表时间：
2006-02-21
期刊：
影响因子：
2.9
通讯作者：
Fitzpatrick, PF
中科院分区：
文献类型：
--
作者：
Frantom, PA;Seravalli, J;Fitzpatrick, PF
酪氨酸羟基酶(TyrH)是一种依赖蝶呤的酶，催化酪氨酸羟化生成二羟基苯丙氨酸。活性部位铁原子的氧化状态在酶的调节中起着核心作用。用厌氧停流光谱仪测定了几种还原剂还原酪氨酸铁的动力学。厌氧快速冷冻-淬火EPR证实，添加还原剂后TyrH的近紫外光吸光度的变化对应于铁的还原。四氢生物蝶呤对野生型酪氨酸H的还原遵循简单的二级反应机制，其速率常数为2.8+/-0.1mM(-1)，S(-1)。6-甲基四氢蝶呤还原铁酶的二级速率常数为6.1+/-0.1mM(-1)，动力学方程为S(-1)。没有检测到自由基中间体的EPR信号。抗坏血酸、谷胱甘肽和1，4-苯喹都能还原酪氨酸铁，但比四氢生物蝶呤的还原速度慢得多，表明蝶呤是一种生理性的还原剂。E332ATyrH在催化反应中对四氢蝶呤的K-m升高，被四氢蝶呤还原，其动力学参数与野生型酶相同，表明BH4在还原过程中不结合在催化构象中。分子氧对酪氨酸亚铁的氧化反应可描述为一步二级反应，反应速率常数为210 mm(-1)S(-1)。S40E TyrH模拟酶的磷酸化状态，具有与野生型酶相似的氧化和还原动力学，表明磷酸化不直接调节铁和铁的相互转化。
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-benzoqui none 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 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.