CRYSTAL-STRUCTURES OF ACONITASE WITH ISOCITRATE AND NITROISOCITRATE BOUND
CRYSTAL-STRUCTURES OF ACONITASE WITH ISOCITRATE AND NITROISOCITRATE BOUND
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DOI：
10.1021/bi00125a014
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发表时间：
1992-03-17
期刊：
影响因子：
2.9
通讯作者：
STOUT, CD
中科院分区：
文献类型：
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作者：
LAUBLE, H;KENNEDY, MC;STOUT, CD
对线粒体异柠檬酸和亚硝基异柠檬酸结合的乌头酸酶的晶体结构进行了解析和修正，在8.02.1埃范围内分别得到R因子为0.179和0.161。用猪心肌酶测定了异柠檬酸结合的结构。穆斯堡尔谱证实了晶体中异柠檬酸的存在。用牛心酶测定了反应中间体亚硝基异柠檬酸结合物的结构。该抑制剂与酶的结合方式几乎与异柠檬酸完全相同。这两种化合物都通过一个羟基氧和一个羧基氧与[4Fe-4S]团簇中唯一的Fe原子结合。一个H2O分子也被束缚，使铁成为六配位的。与它在对称连接的[4Fe-4S]团簇中所处的位置相比，唯一的Fe被从立方体的拐角拉出大约0.2埃。来自乌头酸酶所有四个结构域的至少23个残基参与了活性位点的形成。这些残基参与底物识别(Arg447、Arg452、Arg580、Arg644、Gln72、Ser166、Ser643)、簇连接和相互作用(Cys358、Cys421、Cys424、Asn258、Asn446)以及支持活性位点侧链的氢键(Ala74、Asp568、Ser571、Tbr567)。参与催化的残基是Ser642和三个组氨酸-羧酸对(Asp100-His101，Asp165-His147，Glu262-His167)。从异柠檬酸的C-β中提取质子所需的碱基似乎是Ser642；O-Gamma原子位于计算的氢位置附近，而O-Gamma的环境表明烷氧基(由Arg644的酰胺和侧链形成的氧阴离子空穴)是稳定的。组氨酸-羧酸对似乎是涉及两个氧与铁结合的质子转移反应所必需的，一个来自溶剂(结合水)，另一个来自底物羟基。每个氧都与组氨酸接触，两个氧都与Asp165的侧链接触，Asp165连接了六坐标Fe上的两个位点。
The crystal structures of mitochondrial aconitase with isocitrate and nitroisocitrate bound have been solved and refined to R factors of 0.179 and 0.161, respectively, for all observed data in the range 8.0-2.1 angstrom. Porcine heart enzyme was used for determining the structure with isocitrate bound. The presence of isocitrate in the crystals was corroborated by Mossbauer spectroscopy. Bovine heart enzyme was used for determining the structure with the reaction intermediate analogue nitroisocitrate bound. The inhibitor binds to the enzyme in a manner virtually identical to that of isocitrate. Both compounds bind to the unique Fe atom of the [4Fe-4S] cluster via a hydroxyl oxygen and one carboxyl oxygen. A H2O molecule is also bound, making Fe six-coordinate. The unique Fe is pulled away approximately 0.2 angstrom from the corner of the cubane compared to the position it would occupy in a symmetrically ligated [4Fe-4S] cluster. At least 23 residues from all four domains of aconitase contribute to the active site. These residues participate in substrate recognition (Arg447, Arg452, Arg580, Arg644, Gln72, Ser166, Ser643), cluster ligation and interaction (Cys358, Cys421, Cys424, Asn258, Asn446), and hydrogen bonds supporting active site side chains (Ala74, Asp568, Ser571, Tbr567). Residues implicated in catalysis are Ser642 and three histidine-carboxylate pairs (Asp100-His101, Asp165-His147, Glu262-His167). The base necessary for proton abstraction from C-beta of isocitrate appears to be Ser642; the O-gamma atom is proximal to the calculated hydrogen position, while the environment of O-gamma suggests stabilization of an alkoxide (an oxyanion hole formed by the amide and side chain of Arg644). The histidine-carboxylate pairs appear to be required for proton transfer reactions involving two oxygens bound to Fe, one derived from solvent (bound H2O) and one derived from substrate hydroxyl. Each oxygen is in contact with a histidine, and both are in contact with the side chain of Asp165, which bridges the two sites on the six-coordinate Fe.