Studies on the Synthesis of the Fe-S Cluster of Dihydroxy-acid Dehydratase in Escherichia coli Crude Extract
Studies on the Synthesis of the Fe-S Cluster of Dihydroxy-acid Dehydratase in Escherichia coli Crude Extract
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大肠杆菌粗提物二羟酸脱水酶Fe-S簇的合成研究
DOI：
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发表时间：
1996
影响因子：
4.8
通讯作者：
T. J. Miller
中科院分区：
文献类型：
--
作者：
D. Flint;J. F. Tuminello;T. J. Miller
以大肠杆菌二羟基酸脱水酶的脱辅基蛋白为底物，研究了铁-S簇的合成。失活的辅蛋白在体外可以被大肠杆菌粗提物中的因子重新激活，而在Fe3+、S2−和二硫苏糖醇的存在下，激活的程度要小得多。这种再活化是Fe-S团簇合成的结果。推测在体外粗提物中观察到的铁-S簇合成可能涉及体内参与铁-S簇合成的一些成分。研究了用于形成Fe-S团簇的硫的来源。在大肠杆菌粗提物中发现了四种酶活性，它们可以通过动员半胱氨酸中的硫来为体外铁-S簇合提供硫。本文报道了对其中三种活性负责的蛋白质的纯化。这三种蛋白质分别被鉴定为O-乙酰丝氨酸巯基酶A、O-乙酰丝氨酸巯基酶B和β-胱硫氨酸酶。在O-乙酰丝氨酸巯基酶A和B催化的反应中，从半胱氨酸中动员硫化物的速度和程度取决于亲核剂的存在，这些核试剂可以添加到半胱氨酸脱除硫化物后在酶上形成的氨基丙烯酸酯中。当亲核试剂加到氨基丙烯酸酯上时，就形成了一种新的氨基酸。β-胱硫醚酶不需要亲核剂，反应产物为丙酮酸、氨和硫化物，对β-胱硫醚的裂解反应影响不大。一旦硫被这些酶动员起来，其在铁-S簇合成中的有效利用似乎受到粗提物中存在的未知因素的影响。在存在这些因素的大肠杆菌粗提物和部分纯化制剂中，铁-S簇形成的速度和硫的使用效率意味着铁-S簇的有序控制形成，这通常以酶反应为典型。
The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential [4Fe-4S] cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis. The inactive apoprotein could be reactivated in vitro by factors present in the crude extract of E. coli and to a much smaller extent in the presence of Fe3+, S2−, and dithiothreitol. This reactivation occurs as a result of Fe-S cluster synthesis. It is anticipated that the Fe-S cluster synthesis observed in crude extracts in vitro may involve some of the components that participate in Fe-S cluster synthesis in vivo. The origin of the sulfur used to form Fe-S clusters was investigated. Four enzymatic activities in the crude extract of E. coli were found that can provide sulfur for Fe-S cluster synthesis in vitro by mobilizing the sulfur from cysteine. The purification of the proteins responsible for three of these activities is reported in this paper. The three proteins have been identified as O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B, and β-cystathionase. The rate and extent of sulfide mobilization from cysteine in the reaction catalyzed by O-acetylserine sulfhydrylases A and B depend on the presence of nucleophiles that can add to the aminoacrylate formed on the enzyme following the removal of sulfide from cysteine. A new amino acid is formed when the nucleophiles add to the aminoacrylate. Sulfur mobilization by β-cystathionase does not require a nucleophile, and the reaction is a minor variation on the cleavage of β-cystathionine, with pyruvate, ammonia, and sulfide being the products. Once sulfur is mobilized by these enzymes, its efficient use in Fe-S cluster synthesis seems to be affected by the presence of yet unidentified factors present in crude extract. In crude extract and partially purified preparations from E. coli where these factors are present, the rapidity with which Fe-S clusters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typified by enzymatic reactions.
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影响因子：
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作者：
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DOI：
10.1002/9780470123126.ch1
发表时间：
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期刊：
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影响因子：
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通讯作者：
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DOI：
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发表时间：
1993-07
期刊：
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影响因子：
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作者：
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DOI：
10.1073/pnas.86.10.3639
发表时间：
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影响因子：
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作者：
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通讯作者：
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DOI：
--
发表时间：
1988
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Kennedy,MC;Beinert,H
通讯作者：
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