17O, 1H, and 2H electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the [4Fe-4S]+ cluster of aconitase.
17O, 1H, and 2H electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the [4Fe-4S]+ cluster of aconitase.
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溶剂、底物和抑制剂与乌头酸酶 [4Fe-4S] 簇结合的 17O、1H 和 2H 电子核双共振表征。
DOI：
10.1021/bi00498a015
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Hoffman,BM
中科院分区：
文献类型：
--
作者：
Werst,MM;Kennedy,MC;Beinert,H;Hoffman,BM
西北大学化学系，埃文斯顿，伊利诺伊州60208，以及威斯康星州医学院生物化学系和国家生物医学ESR中心，密尔沃基，威斯康星州53226接收日期：1990年1月31日;修订版Mandarin pt接收日期：1990年6月22日摘要：在X波段（9-GHz）和Q波段（35-GHz）微波频率下的170电子核双共振（ENDOR）研究表明，无底物乌头酸酶[柠檬酸（异柠檬酸）水解酶，EC 4.2. 1.3]结合溶剂HxO（x= 1，2）。既往170项ENDOR研究[Telser et al.（1986）J.Biol.Chem.261，4840-4846]已经公开了HxnO与酶-底物复合物结合，并且还与酶与底物类似物反乌头酸酯和硝基异柠檬酸酯（1-羟基-2-硝基-1，3-丙二羧酸酯）的复合物结合。我们已经使用和2 H ENDOR来表征这些溶剂种类。我们建议，在无底物的酶的第四个配体的Fea是从溶剂中的氢氧根离子，在此Fea网站的底物或底物类似物的结合后，溶剂物种成为质子化，形成一个水分子。先前的170和13 C ENDOR研究[Kennedy et al.等人（1987）Proc. Acad. Sci.我们A. 84，8854-8858]表明，在间乌头酸的丙烷骨架的C-2处或在抑制剂硝基异柠檬酸的Cl处，只有单个羧基与簇配位。总之，这些结果意味着酶催化的柠檬酸和异柠檬酸的相互转化不涉及内源性第四配体的置换，而是添加了阴离子羧酸配体和与Fea结合的溶剂物种的质子化状态的变化。我们进一步报告了170个超精细张量参数的C-2羧基氧的基板绑定到集群确定的170 ENDOR信号的场依赖性。I70 ENDOR研究还表明，抑制剂乌头酸酯的羧基与底物的羧基结合类似。乌头酸酶[柠檬酸（异柠檬酸）水解酶，EC 4.2。1.3]催化柠檬酸盐和异柠檬酸盐经由脱水中间体间乌头酸盐的立体特异性相互转化。
Department of Chemistry, Northwestern University, Evanston, Illinois 60208, and Department of Biochemistry and National Biomedical ESR Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 Received January 31, 1990; Revised Manuscript Received June 22, 1990 abstract: 170 electron nuclear double resonance(ENDOR) studies at X-band (9-GHz) and Q-band (35-GHz) microwave frequencies reveal that the [4Fe-4S]+ cluster of substrate-free aconitase [citrate (isocitrate) hydro-lyase, EC 4.2. 1.3] bindssolvent, HxO {x= 1, 2). Previous 170 ENDOR studies [Telser et al.(1986) J. Biol. Chem. 261, 4840-4846] had disclosed thatHxnO binds to the enzyme-substrate complex and also to complexes of enzyme with the substrate analogues tranj-aconitate and nitroisocitrate (1-hydroxy-2-nitro-1, 3-propanedicarboxylate). We have used and 2H ENDOR to characterize these solvent species. We propose that the fourth ligand of Fea in substrate-free enzyme is a hydroxyl ion from the solvent; upon binding of substrate or substrate analogues at this Fea site, the solvent species becomes protonated to form a water molecule. Previous 170 and 13C ENDOR studies [Kennedy et al.(1987) Proc. Natl. Acad. Sci. US. A. 84, 8854-8858] showed that only a single carboxyl, at C-2 of the propane backbone of m-aconitate or at Cl of the inhibitor nitroisocitrate, coordinates to the cluster. Together, these results imply that enzyme-catalyzed interconversion of citrate and isocitrate does notinvolve displacement of an endogenous fourth ligand, but rather addition of the anionic carboxylate ligand and a change in protonation state of a solvent species bound to Fea. We further report the 170 hyperfine tensor parameters of the C-2 carboxyl oxygen of substrate bound to the cluster as determined by the field dependence of the 170 ENDOR signals. I70 ENDOR studies also show that the carboxyl group of the inhibitor--aconitate binds similarly to that of substrate. e enzyme aconitase [citrate (isocitrate) hydro-lyase, EC 4.2. 1.3] catalyzes the stereospecific interconversion of citrate and isocitrate via the dehydrated intermediate m-aconitate.