The role of intersubunit interactions for the stabilization of the T state of Escherichia coli aspartate transcarbamoylase
The role of intersubunit interactions for the stabilization of the T state of Escherichia coli aspartate transcarbamoylase
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DOI：
10.1074/jbc.m208919200
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发表时间：
2002-12-20
影响因子：
4.8
通讯作者：
Kantrowitz, ER
中科院分区：
文献类型：
--
作者：
Chan, RS;Sakash, JB;Kantrowitz, ER
大肠杆菌天冬氨酸转氨甲酰酶中的同向协同性是由底物诱导的从T态到R态的转变引起的。这两个交替的状态是稳定的一系列域间和亚基间的相互作用。调节链的Lys-143和催化链的Asp-236之间的盐连接仅在T状态下观察到。当Asp-236被丙氨酸取代时，所产生的酶表现出完全的活性，对天冬氨酸的亲和力增强，没有协同性，也没有异嗜性相互作用。这些特征与锁定在功能性R状态的酶一致。使用小角X-射线散射，D236 A突变体的结构后果进行了表征。未配体的D236 A全酶似乎处于一种新的结构状态，既不是T，R，也不是T和R状态的混合物。天然D236 A全酶的结构类似于先前报道的另一种突变体全酶（E239 Q），也缺乏亚基间的相互作用。还研究了天冬氨酸转氨甲酰酶的一个混合版本，其中一个催化亚基是野生型的，另一个具有D236 A突变。混合全酶，与三个六种可能的相互作用，涉及天冬氨酸-236，表现出同向协同性，和异向相互作用与T和R功能状态的酶一致。未连接的杂交的小角X射线散射分析表明，该酶是在一个新的结构状态更类似于T比野生型酶的R状态。这些数据表明，涉及D236 A的六个亚基间相互作用中的三个足以稳定酶的T样状态并允许变构转变。
Homotropic cooperativity in Escherichia coli aspartate transcarbamoylase results from the substrate-induced transition from the T to the R state. These two alternate states are stabilized by a series of interdomain and intersubunit interactions. The salt link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is only observed in the T state. When Asp-236 is replaced by alanine the resulting enzyme exhibits full activity, enhanced affinity for aspartate, no cooperativity, and no heterotropic interactions. These characteristics are consistent with an enzyme locked in the functional R state. Using small angle x-ray scattering, the structural consequences of the D236A mutant were characterized. The unliganded D236A holoenzyme appears to be in a new structural state that is neither T, R, nor a mixture of T and R states. The structure of the native D236A holoenzyme is similar to that previously reported for another mutant holoenzyme (E239Q) that also lacks intersubunit interactions. A hybrid version of aspartate transcarbamoylase in which one catalytic subunit was wild-type and the other had the D236A mutation was also investigated. The hybrid holoenzyme, with three of the six possible interactions involving Asp-236, exhibited homotropic cooperativity, and heterotropic interactions consistent with an enzyme with both T and R functional states. Small angle x-ray scattering analysis of the unligated hybrid indicated that the enzyme was in a new structural state more similar to the T than to the R state of the wild-type enzyme. These data suggest that three of the six intersubunit interactions involving D236A are sufficient to stabilize a T-like state of the enzyme and allow for an allosteric transition.