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首页> 外文期刊>The Biochemical Journal >Characterization of the interactions between Asp(141) and Phe(236) in the Mn2+-L-malate binding of pigeon liver malic enzyme
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Characterization of the interactions between Asp(141) and Phe(236) in the Mn2+-L-malate binding of pigeon liver malic enzyme

机译:鸽肝苹果酸酶Mn2 + -L-苹果酸结合中Asp(141)和Phe(236)之间相互作用的表征

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摘要

The cytosolic malic enzyme from pigeon liver is very sensitive to the metal-catalysed oxidation systems. Our previous studies using the Cu2+-ascorbate as the oxidation system showed that the enzyme was oxidized and cleaved at several positions, including Asp(141). The recently resolved crystal structure of pigeon liver malic enzyme revealed that Asp(141) was near to the metal-binding site, but was not a direct metal ligand. However, Asp(141) is located next to Phe(236), which directly follows the metal ligands Glu(234) and Asp(235). Mutation at Asp(141) caused a drastic effect on the metal-binding affinity of the enzyme. Since Asp(141) and Phe(236) are highly conserved in most species of malic enzyme, we used a double-mutant cycle to study the possible interactions between these two residues. Four single mutants [D141A (Asp(141) --> Ala), D141N, F236A and F236L] and four double mutants (D141A/F236A, D141N/F236A, D141A/F236L and D141N/F236L), plus the wild-type enzyme were successfully cloned, expressed and purified to homogeneity. The secondary, tertiary and quaternary structures of these mutants, as assessed by CD, fluorescence and analytical ultracentrifuge techniques, were similar to that of the wild-type enzyme. Initial velocity experiments were performed to derive the various kinetic parameters, which were used to analyse further the free energy change and the coupling energy (DeltaDeltaG(int)) between any two residues. The dissociation constants for Mn2+ of the D141A and F236A mutants were increased by approx. 6- and 65-fold respectively, compared with that of the wild-type enzyme. However, the K-d.Mn for the double mutant D141A/F236A was only increased by 150-fold. A coupling energy of -2.12 kcal/mol was obtained for Asp(141) and Phe(236). We suggest that Asp(141) is involved in the second sphere of the metal-binding network of the enzyme. [References: 26]
机译:鸽子肝脏的胞质苹果酸酶对金属催化的氧化系统非常敏感。我们先前使用抗坏血酸铜作为氧化系统的研究表明,该酶在包括Asp(141)在内的多个位置被氧化和裂解。鸽肝苹果酸酶的最新解析晶体结构表明,Asp(141)靠近金属结合位点,但不是直接的金属配体。但是,Asp(141)紧邻Phe(236),Phe(236)直接跟随金属配体Glu(234)和Asp(235)。 Asp(141)处的突变对酶的金属结合亲和力产生了巨大影响。由于Asp(141)和Phe(236)在大多数苹果酶物种中高度保守,因此我们使用双突变周期研究了这两个残基之间可能的相互作用。四个单突变体[D141A(Asp(141)-> Ala),D141N,F236A和F236L]和四个双突变体(D141A / F236A,D141N / F236A,D141A / F236L和D141N / F236L),加上野生型酶成功克隆,表达并纯化至同质。通过CD,荧光和分析超速离心技术评估,这些突变体的二级,三级和四级结构与野生型酶相似。进行初始速度实验以导出各种动力学参数,这些动力学参数用于进一步分析任意两个残基之间的自由能变化和耦合能(DeltaDeltaG(int))。 D141A和F236A突变体的Mn2 +的解离常数增加了约1。分别是野生型酶的6倍和65倍。然而,双突变体D141A / F236A的K-d.Mn仅增加了150倍。 Asp(141)和Phe(236)的耦合能为-2.12 kcal / mol。我们建议Asp(141)参与酶的金属结合网络的第二个领域。 [参考:26]

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