Mechanism of flavin reduction in class 2 dihydroorotate dehydrogenases
Mechanism of flavin reduction in class 2 dihydroorotate dehydrogenases
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DOI：
10.1021/bi060919g
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发表时间：
2006-12-19
期刊：
影响因子：
2.9
通讯作者：
Palfey, Bruce A.
中科院分区：
文献类型：
--
作者：
Fagan, Rebecca L.;Nelson, Maria N.;Palfey, Bruce A.
二氢乳清酸脱氢酶（DHOD）使用FMN辅基将二氢乳清酸（DHO）氧化为乳清酸，以从C6和蛋白质残基（对于2类DHOD为Ser）提取氢化物等价物，从而使C5去质子化。两个DHO的C-H键的断裂是否是协调或逐步解决的基本问题2类酶，那些从大肠杆菌和智人，通过确定动力学同位素对黄素还原在厌氧停流实验。测定了E.大肠杆菌酶在低于先前报道的pK（a）控制还原的两个pH值下的反应[Palestine，B.一、Bjornberg，O.，和詹森K. F.（2001）Biochemistry 40，4381-4390]，并且对于在5位标记的DHO为约3倍，对于在6位标记的DHO为约4倍，并且对于在5位和6位两者标记的DHO为约6-7倍。这些同位素效应与DHO的逐步氧化或具有显著量子力学隧穿的协同机制一致。当pH值高于pKa控制还原时，在E. coli DHOD中的5位氘代DHO（反应中的质子供体）。这与逐步反应一致;在（动力学）pKa以上，C5的去质子化足够快，以至于它对观察到的速率常数没有贡献，因此对同位素不敏感。所有可用的信息都指向Ser作为质子中继网络中的一个组件，该网络允许其瞬时去质子化。螺杆sapiens DHOD似乎也具有接近9.4的pKa控制降低，类似于先前报道的E.大肠杆菌酶。用H. sapiens酶在低于pKa的pH值。
Dihydroorotate dehydrogenases (DHODs) oxidize dihydroorotate (DHO) to orotate using the FMN prosthetic group to abstract a hydride equivalent from C6 and a protein residue (Ser for Class 2 DHODs) to deprotonate C5. The fundamental question of whether the scission of the two DHO C-H bonds is concerted or stepwise was addressed for two Class 2 enzymes, those from Escherichia coli and Homo sapiens, by determining kinetic isotope effects on flavin reduction in anaerobic stopped-flow experiments. Isotope effects were determined for the E. coli enzyme at two pH values below a previously reported pK(a) controlling reduction [Palfey, B. A., Bjornberg, O., and Jensen K. F. (2001) Biochemistry 40, 4381-4390] and were about 3-fold for DHO labeled at the 5-position, about 4-fold for DHO labeled at the 6-position, and about 6-7-fold for DHO labeled at both the 5- and 6-positions. These isotope effects are consistent with either a stepwise oxidation of DHO or a concerted mechanism with significant quantum mechanical tunneling. At a pH value above the pKa controlling reduction, no isotope effect was observed in E. coli DHOD for DHO deuterated at the 5- position (the proton donor in the reaction). This is consistent with a stepwise reaction; above the (kinetic) pKa, the deprotonation of C5 is fast enough that it does not contribute to the observed rate constant and, therefore, is not isotopically sensitive. All available information points to Ser acting as a component in a proton relay network which allows its transient deprotonation. The H. sapiens DHOD also appears to have a pKa near 9.4 controlling reduction, similar to that previously reported for the E. coli enzyme. Similar KIEs were obtained with the H. sapiens enzyme at a pH value below the pKa.