Chemical and kinetic reaction mechanisms of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans.
Chemical and kinetic reaction mechanisms of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans.
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DOI：
10.1021/bi035062r
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发表时间：
2003-09
期刊：
影响因子：
2.9
通讯作者：
Dapeng Sun;K. Ono;T. Okajima;K. Tanizawa;M. Uchida;Yukio Yamamoto*;F. Mathews;V. Davidson
中科院分区：
文献类型：
--
作者：
Dapeng Sun;K. Ono;T. Okajima;K. Tanizawa;M. Uchida;Yukio Yamamoto*;F. Mathews;V. Davidson
奎诺血红素蛋白胺脱氢酶（QHNDH）具有催化伯胺的氧化脱氨基的半胱氨酸奎诺基醌（CTQ）辅基。除了CTQ之外，QHNDH中还存在两个血红素c辅因子，其介导底物衍生的电子从CTQ转移到外部电子受体。稳态动力学分析得到相对较小的k（cat）值（<6 s（-1）），速率限制步骤似乎是从QHNDH中的血红素到外部电子受体的蛋白质间电子转移。对QHNDH中血红素通过胺底物的CTQ依赖性还原的瞬态动力学研究对于不同的底物产生了不同的速率常数（对于甲胺、丁胺和苄胺分别为72、190和162 s（-1））。氘动力学同位素效应（KIE）值分别为5.3，3.9和8.5，观察到的甲胺，丁胺，和苄胺的反应。这些结果表明，从α-亚甲基基团的基板，这是伴随着CTQ还原发生的质子的抽象，是在QHNDH的血红素的CTQ依赖性还原这些胺底物的限速步骤。与此相反，2-苯乙胺与QHNDH的反应没有表现出显著的KIE（（H）k（3）/（D）k（3）= 1.05），并且表现出小得多的速率常数16 s（-1）。这表明，对于2-苯乙胺，在单周转反应中的限速步骤是水解的亚胺反应中间体从CTQ或产品释放前的蛋白质内电子转移。QHNDH与手性氘代2-苯基乙胺反应的产物的分析表明，该酶以高度立体特异性的方式提取底物的pro-S质子。苯肼抑制QHNDH的晶体结构的检查表明，Asp 33（γ）是执行质子提取的残基。这些结果的基础上，QHNDH的动力学和化学反应机制提出和讨论的背景下的晶体结构的酶。
Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines. In addition to CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ to an external electron acceptor. Steady-state kinetic assays yielded relatively small k(cat) values (<6 s(-1)), and the rate-limiting step appears to be the interprotein electron transfer from heme in QHNDH to the external electron acceptor. Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s(-1) for methylamine, butylamine, and benzylamine, respectively). Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine. These results suggest that the abstraction of a proton from the alpha-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates. In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ((H)k(3)/(D)k(3) = 1.05) and exhibits a much smaller rate constant of 16 s(-1). This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction is either hydrolysis of the imine reaction intermediate from CTQ or product release prior to intraprotein electron transfer. Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner. Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33(gamma) is the residue that performs the proton abstraction. On the basis of these results, kinetic and chemical reaction mechanisms for QHNDH are proposed and discussed in the context of the crystal structure of the enzyme.