Pavon Jorge Alex, Fitzpatrick Paul F
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Biochemistry. 2006 Sep 12;45(36):11030-7. doi: 10.1021/bi0607554.
Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.
苯丙氨酸羟化酶（PheH）和色氨酸羟化酶（TrpH）分别催化苯丙氨酸和色氨酸的芳香族羟化反应，生成酪氨酸和5-羟色氨酸。以[4-(2)H]-、[3,5-(2)H(2)]-和(2)H(5)-苯丙氨酸为底物研究了PheH和TrpH的反应。大鼠苯丙氨酸羟化酶的催化核心Delta117PheH的所有（D）k(cat)值均正常，范围为1.12 - 1.41。相比之下，对于Delta117PheH V379D（一种四氢生物蝶呤氧化与氨基酸羟化化学计量比发生改变的突变蛋白），[4-(2)H]-苯丙氨酸的（D）k(cat)值为0.92，但[3,5-(2)H(2)]-苯丙氨酸的该值正常。Delta117PheH V379D的四氢生物蝶呤氧化与氨基酸羟化的比率对[4-(2)H]-苯丙氨酸显示出类似的逆同位素效应。由[4-(2)H]-和[3,5(2)H(2)]-苯丙氨酸生成的酪氨酸的氘含量所确定的分子内同位素效应对于Delta117PheH和Delta117PheH V379D是相同的，这表明在突变蛋白中氧添加后的步骤未受影响。这些逆效应与在氨基酸侧链对位的活化铁氧物种反应形成阳离子中间体一致。野生型酶对（D）k(cat)值的正常效应归因于对二烯酮中间体向酪氨酸互变异构化的5.1倍同位素效应，其速率常数是羟化反应的6至7倍。此外，对于最初在苯丙氨酸C4位的氢的损失存在轻微（约34%）偏好。以(2)H(5)-吲哚 - 色氨酸作为Delta117PheH的底物时，（D）k(cat)值为0.89，这与在这种情况下羟化是限速步骤一致。当用氘代苯丙氨酸作为TrpH的底物时，（D）k(cat)值在Delta117PheH V379D的误差范围内。总体而言，这些结果与芳香族氨基酸羟化酶都共享相同的化学机制一致，但PheH羟化的同位素效应被烯二酮中间体向酪氨酸的互变异构所掩盖。
