Protein engineering of pyruvate carboxylase: investigation on the function of acetyl-CoA and the quaternary structure.

Shinji Sueda, Md Nurul Islam, Hiroki Kondo
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引用次数: 25

Abstract

Pyruvate carboxylase (PC) from Bacillus thermodenitrificans was engineered in such a way that the polypeptide chain was divided into two, between the biotin carboxylase (BC) and carboxyl transferase (CT) domains. The two proteins thus formed, PC-(BC) and PC-(CT+BCCP), retained their catalytic activity as assayed by biotin-dependent ATPase and oxamate-dependent oxalacetate decarboxylation, for the former and the latter, respectively. Neither activity was dependent on acetyl-CoA, in sharp contrast to the complete reaction of intact PC. When assessed by gel filtration chromatography, PC-(BC) was found to exist either in dimers or monomers, depending on the protein concentration, while PC-(CT + BCCP) occurred in dimers for the most part. The two proteins do not associate spontaneously or in the presence of acetyl-CoA. Based on these observations, this paper discusses how the tetrameric structure of PC is built up and how acetyl-CoA modulates the protein structure.

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丙酮酸羧化酶的蛋白质工程:乙酰辅酶a的功能及其四级结构的研究。
对热反硝化芽孢杆菌的丙酮酸羧化酶(PC)进行了工程设计,使其多肽链在生物素羧化酶(BC)和羧基转移酶(CT)结构域之间分为两个结构域。由此形成的两种蛋白PC-(BC)和PC-(CT+BCCP)分别通过生物素依赖的atp酶和草酸依赖的草酸脱羧检测保持了它们的催化活性。这两种活性都不依赖于乙酰辅酶a,与完整PC的完全反应形成鲜明对比。通过凝胶过滤层析分析,发现PC-(BC)存在于二聚体或单体中,这取决于蛋白质浓度,而PC-(CT + BCCP)大部分存在于二聚体中。这两种蛋白质不能自发结合或在乙酰辅酶a存在下结合。在此基础上,本文讨论了PC的四聚体结构是如何建立的,以及乙酰辅酶a是如何调节蛋白质结构的。
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