Fagan Rebecca L, Nelson Maria N, Pagano Paul M, Palfey Bruce A
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
Biochemistry. 2006 Dec 19;45(50):14926-32. doi: 10.1021/bi060919g.
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 pKa controlling reduction [Palfey, B. A., Björnberg, 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.
二氢乳清酸脱氢酶（DHODs）利用黄素单核苷酸辅基从C6提取一个氢负离子等价物，并利用一个蛋白质残基（2类DHODs中的丝氨酸）使C5去质子化，从而将二氢乳清酸（DHO）氧化为乳清酸。通过在厌氧停流实验中测定黄素还原的动力学同位素效应，研究了两种2类酶（来自大肠杆菌和智人的酶）中DHO的两个C-H键断裂是协同还是分步的基本问题。在两个低于先前报道的控制还原的pKa的pH值下，测定了大肠杆菌酶的同位素效应[帕尔菲，B.A.，比约恩伯格，O.，和延森，K.F.（2001年）《生物化学》40，4381 - 4390]，5位标记的DHO的同位素效应约为3倍，6位标记的DHO的同位素效应约为4倍，5位和6位都标记的DHO的同位素效应约为6 - 7倍。这些同位素效应与DHO的分步氧化或具有显著量子力学隧穿的协同机制一致。在高于控制还原的pKa的pH值下，大肠杆菌DHOD中5位氘代的DHO（反应中的质子供体）未观察到同位素效应。这与分步反应一致；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。所有现有信息都表明丝氨酸作为质子传递网络的一个组成部分，允许其瞬时去质子化。智人的DHOD似乎也有一个接近9.4的控制还原的pKa，与先前报道的大肠杆菌酶相似。在低于pKa的pH值下，智人酶也获得了类似的动力学同位素效应。
