Allostery in rabbit pyruvate kinase: Development of a strategy to elucidate the mechanism
Allostery in rabbit pyruvate kinase: Development of a strategy to elucidate the mechanism
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DOI：
10.1021/bi981273y
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发表时间：
1998-11-03
期刊：
影响因子：
2.9
通讯作者：
Braun, W
中科院分区：
文献类型：
--
作者：
Friesen, RHE;Castellani, RJ;Braun, W
丙酮酸激酶（PK）同工酶在兔肌肉和肾脏中的表达具有不同的变构动力学。两种同工酶中唯一的氨基酸变化，源于选择性RNA剪接，发生在亚基界面附近的C结构域中的55个氨基酸的延伸处。自校正距离几何（SECODG）程序DIAMOD用于计算肾PK二聚体的四螺旋束中这些界面接触的同源性模型，基于四聚体兔肌肉PK的X-射线结构[Larsen等人（1994）Biochemistry 33，6301-6309]，Energyrefinement with the program FANTOM，使用ECEPP/2力场评估两个亚基之间的堆积和静电相互作用，产生两组能量有利的构象。两组的主要差异是残基Pre 402的环构象，其是肌肉PK中的丝氨酸。在一个环构象中，保守的赖氨酸421可以形成亚基间盐桥，如在肌肉PK晶体结构中观察到的。另一个环构象有利于替代的亚基内盐桥，类似于大肠杆菌PK结构中发现的，其不用于生成模型。亚基间盐桥导致一个亚基的Lys 421和另一个亚基的Tyr 443之间的亚基间氢键。为了提供关于这些残基的作用的直接证据，进行了肌肉PK基因的定点诱变。将Ser 402转化为脯氨酸和Tyr 443转化为苯丙氨酸既不改变二级结构也不改变四聚体结构，如分别通过远紫外-CD和沉降速度测量的。然而，S402 P突变体表现出稳态动力学，表明该突变体是更responsive的调节效应，而突变Y 443 F基本上相当于野生型肌肉PK蛋白，除了较低的亲和力磷酸烯醇丙酮酸。这些结果表明，在PK的变构调节中的一些关键残基的关键作用。
Isozymes of pyruvate kinase (PK) expressed in rabbit muscle and kidney show different allosteric kinetics. The only amino acid changes in the two isozymes, originating from alternative RNA splicing, occur at a stretch of 55 amino acids in the C domain near the subunit interface, The self-correcting distance geometry (SECODG) program DIAMOD was used to calculate a homology model of these interfacial contacts in the four helix bundle of the kidney PK dimer, based on the X-ray structure of the tetrameric rabbit muscle PK [Larsen et al. (1994) Biochemistry 33, 6301-6309], Energy refinement with the program FANTOM, using the ECEPP/2 force field to assess packing and electrostatic interactions between the two subunits, yielded two groups of energetically favorable conformations. The primary difference in the two groups is the loop conformation of residue Pre 402, which is serine in muscle PK. In one loop conformation, the conserved Lys 421 can form an intersubunit salt bridge as observed in the muscle PK crystal structure. The other loop conformation favors an alternative intrasubunit salt bridge, similar to that found in the Escherichia coli PK structure, which was not used for generating the model. The intersubunit salt bridge leads to an intersubunit hydrogen bonding between Lys 421 of one subunit and Tyr 443 of the other. To provide direct evidence on the roles of these residues, site-directed mutagenesis of the muscle PK gene was conducted. Converting Ser 402 to a proline and Tyr 443 to a phenylalanine changed neither the secondary nor the tetrameric structure, as measured by far UV-CD and sedimentation velocity, respectively. However, the S402P mutant exhibits steady-state kinetics, indicating that the mutant is more reponsive to regulation by effecters, while the mutant Y443F was essentially equivalent to wildtype muscle PK protein except for a lower affinity to phosphoenolpyruvate. These findings suggest a pivotal role for a few key residues in the allosteric regulation in PK.