Measurement of the intramolecular isotope effect on aliphatic hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase.
Measurement of the intramolecular isotope effect on aliphatic hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase.
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紫色色杆菌苯丙氨酸羟化酶对脂肪族羟基化的分子内同位素效应的测量。
DOI：
10.1021/ja101563t
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发表时间：
2010
影响因子：
15
通讯作者：
Fitzpatrick,PaulF
中科院分区：
文献类型：
--
作者：
Panay,AramJ;Fitzpatrick,PaulF
紫色杆菌产生的非血红素铁酶苯丙氨酸羟基酶除了催化正常的芳香族羟化反应外，还能催化苯甲酸和脂肪族碳的羟化反应。为了了解铁中心的反应活性，测定了该酶对3-环己基丙氨酸羟化反应的本征同位素效应。以3-[2H11-环己基]丙氨酸为底物，同位素效应对Kcat值的影响为1，与整个反应中的另一个步骤明显慢于羟基化一致。因此，通过测量3-[1，2，3，4，5，6-2H6-环己基]丙氨酸羟化过程中损失的氚的量，确定了同位素效应为分子内效应。保留氢原子的4-HO-环己基丙氨酸与失去一个氢原子的4-HO-环己基丙氨酸之比为2.8。这给出了一个计算值为12.6的比率的一次氚动力学同位素效应与二次同位素效应。这一值与亲电中心Fe(O)对氢原子的抽离和量子力学隧穿对反应的贡献是一致的。
The non-heme iron enzyme phenylalanine hydroxylase from Chromobacterium violaceum has previously been shown to catalyze the hydroxylation of benzylic and aliphatic carbons in addition to the normal aromatic hydroxylation reaction. The intrinsic isotope effect for hydroxylation of 3-cyclochexylalanine by the enzyme was determined in order to gain insight into the reactivity of the iron center. With 3-[2H11-cyclohexyl]alanine as the substrate, the isotope effect on thekcatvalue was 1, consistent with an additional step in the overall reaction being significantly slower than hydroxylation. Consequently, the isotope effect was determined as an intramolecular effect by measuring the amount of deuterium lost in the hydroxylation of 3-[1,2,3,4,5,6-2H6-cyclohexyl]alanine. The ratio of 4-HO-cyclohexylalanine that retained deuterium to that which lost one deuterium atom was 2.8. This gave a calculated value of 12.6 for the ratio of the primary deuterium kinetic isotope effect to the secondary isotope effect. This value is consistent with hydrogen atom abstraction by an electrophilic Fe(O) center and a contribution of quantum-mechanical tunneling to the reaction.