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首页> 外文期刊>Biochemistry >The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase
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The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

机译:部分底物去硫代乙酰辅酶A模拟嗜酸嗜热菌柠檬酸合酶催化的缩合半反应中的所有关键碳酸反应。

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摘要

Citrate synthase (CS) performs two half-reactions: the mechanistically intriguing condensation ofnacetyl-CoA with oxaloacetate (OAA) to form citryl-CoA and the subsequent, slower hydrolysis of citryl-CoAnthat generally dominates steady-state kinetics. The condensation reaction requires the abstraction of a protonnfrom the methyl carbon of acetyl-CoA to generate a reactive enolate intermediate. The carbanion of thatnintermediate then attacks the OAA carbonyl to furnish citryl-CoA, the initial product. Using stopped-flownand steady-state fluorescence methods, kinetic substrate isotope effects, and mutagenesis of active sitenresidues, we show that all of the processes that occur in the condensation half-reaction performed bynThermoplasma acidophilum citrate synthase (TpCS) with the natural thioester substrate, acetyl-CoA, alsonoccur with the ketone inhibitor dethiaacetyl-CoA. Free energy profiles demonstrate that the nonhydrolyzablenproduct of the condensation reaction, dethiacitryl-CoA, forms a particularly stable complex with TpCS butnnot pig heart CS.
机译:柠檬酸合酶(CS)进行两个半反应:机械吸引人的n乙酰-CoA与草酰乙酸(OAA)的缩合反应形成citryl-CoA;随后的citryl-CoAn水解较慢,通常主要控制稳态动力学。缩合反应需要从乙酰辅酶A的甲基碳中提取质子,以生成反应性烯醇中间体。然后,该中间体的碳负离子侵蚀OAA羰基,提供初始产物citryl-CoA。使用停止流动和稳态荧光方法,动力学底物同位素效应以及活性位点残基的诱变,我们显示了嗜酸嗜热单胞菌柠檬酸合酶(TpCS)与天然硫酯底物进行的缩合半反应中发生的所有过程,乙酰辅酶A,也不会与酮抑制剂去硫乙酰辅酶A发生。自由能曲线表明,缩合反应的非水解产物去硫胞嘧啶-CoA与TpCS形成了特别稳定的复合物,但没有猪心CS。

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  • 来源
    《Biochemistry》 |2009年第33期|p.7878-7891|共14页
  • 作者单位

    ‡Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110,§Department of Chemistry, Washington University, St. Louis, Missouri 63130, and)Department of Biochemistry, Purdue University,West Lafayette, Indiana 47907-2063;

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