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首页> 外文期刊>Biochemistry >Evidence for the Mechanism of Hydroxylation by 4-Hydroxyphenylpyruvate Dioxygenase and Hydroxymandelate Synthase from Intermediate Partitioning in Active Site Variants
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Evidence for the Mechanism of Hydroxylation by 4-Hydroxyphenylpyruvate Dioxygenase and Hydroxymandelate Synthase from Intermediate Partitioning in Active Site Variants

机译：4-位羟基苯丙酮酸双加氧酶和羟基扁桃酸合酶由中间位点在活性位点变异中进行羟化反应的证据。

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4-Hydroxyphenylpyruvate dioxygenase (HPPD) and hydroxymandelate synthase (HMS) each catalyze similar complex dioxygenation reactions using the substrates 4-hydroxyphenylpyruvate (HPP) and dioxygen. The reactions differ in that HPPD hydroxylates at the ring C1 and HMS at the benzylic position. The HPPD reaction is more complex in that hydroxylation at C1 instigates a 1,2-shift of an aceto substituent. Despite that multiple intermediates have been observed to accumulate in single turnover reactions of both enzymes, neither enzyme exhibits significant accumulation of the hydroxylating intermediate. In this study we employ a product analysis method based on the extents of intermediate partitioning with HPP deuterium substitutions to measure the kinetic isotope effects for hydroxylation. These data suggest that, when forming the native product homogentisate, the wild-type form of HPPD produces a ring epoxide as the immediate product of hydroxylation but that the variant HPPDs tended to also show the intermediacy of a benzylic cation for this step. Similarly, the kinetic isotope effects for the other major product observed, quinolacetic acid, showed that either pathway is possible. HMS variants show small normal kinetic isotope effects that indicate displacement of the deuteron in the hydroxylation step. The relatively small magnitude of this value argues best for a hydrogen atom abstraction/rebound mechanism. These data are the first definitive evidence for the nature of the hydroxylation reactions of HPPD and HMS.
机译：4-羟基苯基丙酮酸双加氧酶（HPPD）和羟基扁桃酸酯合酶（HMS）各自使用底物4-羟基苯基丙酮酸（HPP）和双氧催化相似的复杂双加氧反应。反应的不同之处在于，HPPD在环C1处羟基化，而HMS在苄基位置处羟基化。 HPPD反应更复杂，因为在C1处的羟基化促使乙酰取代基发生1，2-移位。尽管已观察到多种中间体在两种酶的单次转化反应中积累，但两种酶均未显示出明显的羟基化中间体积累。在这项研究中，我们采用基于HPP氘取代的中间分配程度的产物分析方法来测量羟基的动力学同位素效应。这些数据表明，当形成天然产物尿黑酸时，HPPD的野生型形式产生环氧化物作为羟基化的直接产物，但是该步骤的HPPD变体也倾向于显示苄基阳离子的中间性。同样，观察到的另一种主要产物喹诺乙酸的动力学同位素效应表明，这两种途径都是可能的。 HMS变体显示出较小的正态动力学同位素效应，表明氘化了羟基化步骤中的氘核。该值的相对较小的值最适合氢原子的提取/回弹机制。这些数据是有关HPPD和HMS羟基化反应性质的第一个明确证据。

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