首页> 美国卫生研究院文献>other >Mutagenesis of a Specificity-Determining Residue in Tyrosine Hydroxylase Establishes that the Enzyme is a Robust Phenylalanine Hydroxylase but a Fragile Tyrosine Hydroxylase
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Mutagenesis of a Specificity-Determining Residue in Tyrosine Hydroxylase Establishes that the Enzyme is a Robust Phenylalanine Hydroxylase but a Fragile Tyrosine Hydroxylase

机译:一个特异性决定残留量酪氨酸羟化酶建立的突变这种酶是一种强大的苯丙氨酸羟化酶但脆弱的酪氨酸羟化酶

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摘要

The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites, but PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme (Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry >39, 9652–9661). Alanine scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 104) decreases in the Vmax and Vmax/Ktyr values for tyrosine hydroxylation, but only small decreases or even increases in the Vmax and Vmax/Kphe values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the Vmax value with tyrosine as substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur.
机译:芳香族氨基酸羟化酶酪氨酸羟化酶(TyrH)和苯丙氨酸羟化酶(PheH)具有基本相同的活性位点,但是PheH几乎不能羟化酪氨酸,而TyrH可以轻易地羟化酪氨酸和苯丙氨酸。先前的研究表明,TyrH的Asp425在确定该酶的底物特异性方面很重要(Daubner,S. C.,Melendez,J.和Fitzpatrick,P. F.(2000)生物化学> 39 ,9652–9661)。氨基酸423-427的丙氨酸扫描诱变是一个包含Asp425的移动环,显示只有诱变Asp425才能显着改变酶的活性。 Asp425的饱和诱变会导致酪氨酸羟基化的Vmax和Vmax / Ktyr值大幅下降(最多10 4 ),但苯丙氨酸的Vmax和Vmax / Kphe值仅出现很小的下降甚至上升羟基化。突变蛋白酪氨酸羟化活性的降低是由于四氢蝶呤的氧化与酪氨酸作为氨基酸底物的氨基酸羟化解偶联。相反,除D425W突变体外,四苯蝶呤氧化和氨基酸羟基化的偶联不受影响或以苯丙氨酸为氨基酸底物增加。以酪氨酸为底物的Vmax值的降低与425位氨基酸残基的疏水性呈负相关。结果与Asp425的关键作用相一致,即防止疏水性相互作用导致活性位点受限。不会发生酪氨酸的羟基化。

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