Mutation of serine 395 of tyrosine hydroxylase decouples oxygen-oxygen bond cleavage and tyrosine hydroxylation
Mutation of serine 395 of tyrosine hydroxylase decouples oxygen-oxygen bond cleavage and tyrosine hydroxylation
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DOI：
10.1021/bi9928546
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发表时间：
2000-04-11
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：
Ellis, HR;Daubner, SC;Fitzpatrick, PF
大鼠酪氨酸羟化酶活性位点的Ser 395和Ser 396在蝶呤依赖性羟化酶家族的所有三个成员中是保守的，即苯丙氨酸羟化酶、酪氨酸羟化酶和色氨酸羟化酶。Ser 395被适当地定位以与His 331的咪唑氮形成氢键，His 331是活性位点铁的轴向配体，而Ser 396位于活性位点裂缝的壁上。定点突变已被用来分析这两个残基在催化中的作用。由S395 A、S395 T和S396 A酶形成二羟基苯丙氨酸的比活性分别为野生型值的1.3%、26%和69%。S395 A和S396 A酶都结合化学计量量的铁，并且当与多巴胺复合时表现出野生型光谱。酪氨酸、6-甲基四氢蝶呤和四氢生物蝶呤的K-M值不受用丙氨酸替换任一残基的影响。尽管S395 A酶对酪氨酸羟基化的Vmax值降低了2个数量级，但S395 A或S396 A酶对四氢蝶呤氧化的Vmax值与野生型值相比没有变化。使用这两种突变酶，从6-甲基四氢蝶呤定量形成4 α-羟基蝶呤。这些结果表明，Ser 395是必需的氨基酸羟基化，但不为氧-氧键的切割，而Ser 396不是必需的。这些结果还证实，氧-氧键的断裂发生在与氨基酸羟基化分开的步骤中。
Ser395 and Ser396 in the active site of rat tyrosine hydroxylase are conserved in all three members of the family of pterin-dependent hydroxylases, phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Ser395 is appropriately positioned to form a hydrogen bond to the imidazole nitrogen of His331, an axial ligand to the active site iron, while Ser396 is located on the wall of the active site cleft. Site-directed mutagenesis has been used to analyze the roles of these two residues in catalysis. The specific activities for formation of dihydroxyphenylalanine by the S395A, S395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respectively. Both the S395A and S396A enzymes bind a stoichiometric amount of iron and exhibit wild-type spectra when complexed with dopamine. The K-M values for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are unaffected by replacement of either residue with alanine. Although the V-max value for tyrosine hydroxylation by the S395A enzyme is decreased by 2 orders of magnitude, the V-max value for tetrahydropterin oxidation by either the S395A or the S396A enzyme is unchanged from the wild-type value. With both mutant enzymes, there is quantitative formation of 4a-hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required for amino acid hydroxylation but not for cleavage of the oxygen-oxygen bond, while Ser396 is not essential. These results also establish that cleavage of the oxygen-oxygen bond occurs in a separate step from amino acid hydroxylation.