Mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase increases the affinity for phenylalanine.
Mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase increases the affinity for phenylalanine.
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酪氨酸羟化酶中酪氨酸 371 突变为苯丙氨酸，增加了对苯丙氨酸的亲和力。
DOI：
10.1021/bi981648f
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Fitzpatrick,PF
中科院分区：
文献类型：
--
作者：
Daubner,SC;Fitzpatrick,PF
芳香族氨基酸羟化酶酪氨酸和苯丙氨酸羟化酶都含有非血红素铁，利用氧和四氢生物蝶呤，并且是相同亚基的四聚体。这些酶的催化结构域是同源的，并且最近的X射线晶体学分析显示这两种酶的活性位点非常相似。酪氨酸羟化酶中酪氨酸371和苯丙氨酸羟化酶中酪氨酸325的羟基氧分别距离酶中活性位点铁5和4.5 μ m。为了确定该残基是否如先前所建议的在催化机制中起作用[Erlandsen，H.，等人（1997）Nat. Struct. Biol.4，995 - 1000]，通过定点诱变将酪氨酸羟化酶的酪氨酸371改变为苯丙氨酸。Y371 F蛋白在酪氨酸羟基化中完全活跃，消除了该残基的基本机械作用。苯丙氨酸或4-甲基苯丙氨酸作为底物时，产物分布没有变化，表明羟基化中间体的反应性不受影响。然而，突变蛋白中苯丙氨酸的Km值降低了10倍。这些结果被解释为在突变蛋白的活性位点的更大的构象灵活性的指示。
The aromatic amino acid hydroxylases tyrosine and phenylalanine hydroxylase both contain non-heme iron, utilize oxygen and tetrahydrobiopterin, and are tetramers of identical subunits. The catalytic domains of these enzymes are homologous, and recent X-ray crystallographic analyses show the active sites of the two enzymes are very similar. The hydroxyl oxygens of tyrosine 371 in tyrosine hydroxylase and of tyrosine 325 of phenylalanine hydroxylase are 5 and 4.5 Å, respectively, away from the active site iron in the enzymes. To determine whether this residue has a role in the catalytic mechanism as previously suggested [Erlandsen, H., et al. (1997)Nat.Struct. Biol.4, 995−1000], tyrosine 371 of tyrosine hydroxylase was altered to phenylalanine by site-directed mutagenesis. The Y371F protein was fully active in tyrosine hydroxylation, eliminating an essential mechanistic role for this residue. There was no change in the product distribution seen with phenylalanine or 4-methylphenylalanine as a substrate, suggesting that the reactivity of the hydroxylating intermediate was unaffected. However, theKMvalue for phenylalanine was decreased 10-fold in the mutant protein. These results are interpreted as an indication of greater conformational flexibility in the active site of the mutant protein.