Fe-S centers in lactyl-CoA dehydratase.
Fe-S centers in lactyl-CoA dehydratase.
复制标题
Fe-S 以乳酰辅酶 A 脱水酶为中心。
DOI：
10.1021/bi00371a009
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Abeles,RH
中科院分区：
文献类型：
--
作者：
Kuchta,RD;Hanson,GR;Holmquist,B;Abeles,RH
Robert D. Kuchta, 1 Graeme R. Hanson, 8, 11 Barton Holmquist，*·8和Robert H. Abeles1马萨诸塞州沃瑟姆市布兰萨梅斯大学生物化学系，马萨诸塞州沃瑟姆市02254，哈佛医学院生物化学和生物物理科学与医学中心，布莱根妇女医院，马萨诸塞州波士顿02115,1986年5月21日；摘要：Lactyl-CoA脱水酶由El和E2两种酶组成，需要催化量的ATP才能发挥活性[Kuchta， RD, & Abeles， R. H.(1985) J. Biol。与不含铁的El相比，E2含有8.20±0.04 mol Fe/mol E2，其中1个可以被1,10 -菲罗啉去除。E2还含有7.33±0.68 mol的无机硫/mol的E2，表明至少有7个铁原子以Fe- s簇的形式存在。El和E2的Cu、Co、Zn、Mn和Ni含量低于0.14 mol /mol El或E2。在4k下，还原和氧化的El在10000-G扫描范围内都是EPR沉默的，而在4k下可以观察到E2中的两个信号。含有7个或8个铁原子的E2获得了相同的光谱，但这两种信号仅在T< 30 K时可观察到。信号1轴对称，g±=2.0232,g $= 2.0006。信号2与g正交，= 1.982,g2= 1.995, g3=2.019。利用S′= 1/2自旋哈密顿量对这些光谱进行计算机模拟，提取g矩阵。随着Na2S204对E2的还原，这两个信号的强度减小。我们提出信号1是由一个不寻常的[4Fe-4S]集群引起的，而信号2是由一个[3Fe-3/4S]集群引起的。添加丙烯酰辅酶a或丙烯酰辅酶a可显著改变信号2。因此，底物与E2结合并改变[3Fe-3/4S]簇的环境。ATP和El都不会改变E2的光谱。02使E2失活，导致黄素和[3Fe-3/4S]簇的损失。在提纯E2的过程中加入快速蛋白液相色谱法会导致黄素的部分损失，并改变E2，使Fe-S中心变得更难氧化或还原，但对催化活性的影响要小得多。在乙酰辅酶a脱水过程中，氧化活性中心对E2的作用尚不清楚。
Robert D. Kuchta, 1 Graeme R. Hanson, 8, 11 Barton Holmquist,*· 8 and Robert H. Abeles1 Department of Biochemistry, Brandéis University, Waltham, Massachusetts 02254, and Center for Biochemical and Biophysical Sciences andMedicine, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts 02115 Received May 21, 1986; Revised Manuscript Received July 25, 1986 abstract: Lactyl-CoA dehydratase consists of two enzymes, El and E2, and requires catalytic quantities of ATP for activity [Kuchta, RD, & Abeles, R. H.(1985) J. Biol. Chem. 260, 13181-13189], In contrast to El, which contains no Fe, E2contains 8.20±0.04 mol of Fe/mol of E2, one of which can be removed by 1, 10-phenanthroline. E2 also contains 7.33±0.68 mol of inorganic sulfur/mol of E2, indicating that at least seven of the Fe atoms are present as Fe-S clusters. El and E2 contain< 0.14 mol of Cu, Co, Zn, Mn, and Ni/mol of El or E2. Both reduced and oxidized El are EPR silent over a 10000-G scan range at 4 K, while two signals in E2 are observable at 4 K. Identical spectra were obtained with E2 containing either seven or eight Fe atoms, andboth signals were only observable at T< 30 K. Signal 1 has axial symmetry with g±=2.0232 and g $= 2.0006. Signal 2 is orthorhombic with g,= 1.982, g2= 1.995, and g3=2.019. Computer simulation of these spectra with a S'= 1/2 spin Hamiltonian was used to extract the g matrices. The intensity of both signals decreases when E2 is reduced with Na2S204. We propose that signal 1 is due to an unusual [4Fe-4S] cluster and signal 2 to a [3Fe-3/4S] cluster. Addition of either acrylyl-CoA or lactyl-CoA dramatically alters signal 2. Thus, substrates bind to E2 and alter the environment of the [3Fe-3/4S] cluster. Neither ATP nor El alters the spectrum of E2. 02 inactivates E2 and causes loss of flavin and the [3Fe-3/4S] cluster. Inclusion of fast protein liquid chromatography during purification of E2 causes partial loss of flavin and alters E2 such that the Fe-S centers become more difficult to oxidize or reduce, but has much less effect upon catalytic activity. The role of the redox-active centers on E2 during the dehydration of lactyl-CoA is not known.