Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: Implications for allosteric regulation
Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: Implications for allosteric regulation
复制标题
DOI：
10.1073/pnas.090087197
复制
发表时间：
2000-05-09
影响因子：
11.1
通讯作者：
Schachman, HK
中科院分区：
文献类型：
--
作者：
Endrizzi, JA;Beernink, PT;Schachman, HK
理解酶调节的一个中心问题是确定催化活性变构变化的构象状态。对于大肠杆菌天冬氨酸转氨甲酰酶（ATCase; EC 2.1.3.2），活性的、松弛的（R状态）全酶通常被认为由全酶与双底物类似物N-膦酰基乙酰基-L-天冬氨酸（PALA）的复合物的晶体结构表示。目前还不清楚，但是，其中的构象之间的差异unliganded，无活性，拉紧（T状态）全酶和PALA复合物是由于本地化的影响，抑制剂结合的变构转换相反。为了确定由PALA结合产生的分离的非变构C三聚体的构象变化，我们确定了C三聚体-PALA复合物的1.95埃分辨率晶体结构。与游离C三聚体相反。PALA结合三聚体显示近似三重对称性。PALA结合后C三聚体的构象变化包括两个活性位点环的排序和与N-和C-末端结构域相关的铰链的闭合。C三聚体-PALA结构与PALA结合全酶中的配体C亚基非常相似。这种相似性表明，明显的铰链关闭和其他变化促进PALA结合的全酶是稳定的配体结合。因此，构象变化归因于全酶的变构转换仍然有待确定。
A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. For Escherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-Angstrom resolution crystal structure of the C trimer-PALA complex. In contrast to the free C trimer. the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer-PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.