Uncoupled forms of tyrosine hydroxylase unmask kinetic isotope effects on chemical steps
Uncoupled forms of tyrosine hydroxylase unmask kinetic isotope effects on chemical steps
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DOI：
10.1021/ja0383165
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发表时间：
2003-12-31
影响因子：
15
通讯作者：
Fitzpatrick, PF
中科院分区：
文献类型：
--
作者：
Frantom, PA;Fitzpatrick, PF
酪氨酸羟化酶（TyrH）催化酪氨酸羟基化为二羟基苯丙氨酸。在所提出的机制中，一个ferryl-oxo物种攻击酪氨酸的芳环，形成阳离子中间体。然而，当3，5 - 2 H2-酪氨酸用作底物时，未发现野生型TyrH的显著同位素效应。同位素效应现在已经用3，5 - 2 H2-酪氨酸使用突变形式的TyrH确定，其中蝶呤的氧化与氨基酸的羟基化解偶联。三个突变酶表现出显着的逆氘同位素效应和逆溶剂同位素效应。E326 A酶的质子库存与非生产性步骤上2.4的正常溶剂同位素效应一致。这些结果支持了所提出的机制，并证明了使用具有分支途径的突变蛋白来揭示野生型酶中被掩盖的同位素效应的实用性。
Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine. In the proposed mechanism, a ferryl-oxo species attacks the aromatic ring of tyrosine, forming a cationic intermediate. However, no significant isotope effect is found for wild-type TyrH when 3,5-2H2-tyrosine is used as a substrate. The isotope effect has now been determined with 3,5-2H2-tyrosine using mutant forms of TyrH in which the oxidation of the pterin is uncoupled from hydroxylation of the amino acid. Three mutant enzymes exhibit significant inverse deuterium isotope effects and inverse solvent isotope effects. A proton inventory for the E326A enzyme is consistent with a normal solvent isotope effect of 2.4 on an unproductive step. The results support the proposed mechanism and demonstrate the utility of using mutant proteins with branched pathways to reveal isotope effects which are masked in the wild-type enzyme.