Neidig Michael L, Brown Christina D, Kavana Michael, Choroba Oliver W, Spencer Jonathan B, Moran Graham R, Solomon Edward I
Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
J Inorg Biochem. 2006 Dec;100(12):2108-16. doi: 10.1016/j.jinorgbio.2006.08.021. Epub 2006 Sep 26.
The alpha-ketoglutate (alpha-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require Fe(II), alpha-KG and dioxygen for catalysis, with the alpha-KG cosubstrate supplying the two additional electrons required for dioxygen activation. A sub-class of these enzymes exists in which the alpha-keto acid is covalently attached to the substrate, including (4-hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) which utilize the same substrate but exhibit two different general reactivities (H-atom abstraction and electrophilic attack). Previous kinetic studies of Streptomyces avermitilis HPPD have shown that the substrate analog phenylpyruvate (PPA), which only differs from the normal substrate (4-hydroxyphenyl)pyruvate (HPP) by the absence of a para-hydroxyl group on the aromatic ring, does not induce a reaction with dioxygen. While an Fe(IV)O intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O(2) to generate this species is the decarboxylation of the alpha-keto acid. It has been generally proposed that the two requirements for decarboxylation are bidentate coordination of the alpha-keto acid to Fe(II) and the presence of a 5C Fe(II) site for the O(2) reaction. Circular dichroism and magnetic circular dichroism studies have been performed and indicate that both enzyme complexes with PPA are similar with bidentate alpha-KG coordination and a 5C Fe(II) site. However, kinetic studies indicate that while HmaS reacts with PPA in a coupled reaction similar to the reaction with HPP, HPPD reacts with PPA in an uncoupled reaction at an approximately 10(5)-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n-->pi( *) transition of the HPPD/Fe(II)/PPA complex which, based upon correlation to density functional theory calculations, is suggested to result from H-bonding between a nearby residue and the carboxylate group of the alpha-keto acid. Such an interaction would disfavor the decarboxylation reaction by stabilizing electron density on the carboxylate group such that the oxidative cleavage to yield CO(2) is disfavored.
α-酮戊二酸（α-KG）依赖性双加氧酶是一大类单核非血红素铁酶，催化反应需要Fe（II）、α-KG和氧气，α-KG共底物提供氧气活化所需的另外两个电子。这类酶存在一个亚类，其中α-酮酸与底物共价连接，包括（4-羟基）扁桃酸合酶（HmaS）和（4-羟苯基）丙酮酸双加氧酶（HPPD），它们利用相同的底物，但表现出两种不同的一般反应性（氢原子夺取和亲电攻击）。以往对阿维链霉菌HPPD的动力学研究表明，底物类似物苯丙酮酸（PPA）与正常底物（4-羟苯基）丙酮酸（HPP）的唯一区别是芳环上没有对羟基，它不会引发与氧气的反应。虽然有人提出Fe（IV）O中间体是将底物转化为产物的活性物种，但利用氧气生成该物种的关键步骤是α-酮酸的脱羧反应。一般认为脱羧反应的两个条件是α-酮酸与Fe（II）的双齿配位以及存在用于氧气反应的5配位Fe（II）位点。已经进行了圆二色性和磁圆二色性研究，结果表明与PPA形成的两种酶复合物在α-KG双齿配位和5配位Fe（II）位点方面相似。然而，动力学研究表明，虽然HmaS与PPA的反应类似于与HPP的反应，是偶联反应，但HPPD与PPA的反应是解偶联反应，与HPP反应相比，反应速率降低了约10^5倍。在HPPD/Fe（II）/PPA复合物的n→π*跃迁中通过光谱观察到一个关键差异，基于与密度泛函理论计算的相关性，这被认为是由附近残基与α-酮酸的羧基之间的氢键作用导致的。这种相互作用会通过稳定羧基上的电子密度而不利于脱羧反应，使得氧化裂解生成CO2的反应受到抑制。
