Ligation alters the pathway of urea-induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase.
Ligation alters the pathway of urea-induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase.
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连接改变了尿素诱导的大肠杆菌天冬氨酸转氨甲酰酶催化三聚体变性的途径。
DOI：
10.1002/pro.5560030809
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Allewell,NM
中科院分区：
文献类型：
--
作者：
Bromberg,S;LiCata,VJ;Mallikarachchi,D;Allewell,NM
我们通过分析凝胶色谱法，通过辅以活性的分析凝胶色谱法，在存在和不存在氨甲酰磷酸（CP；底物）、N-(膦乙酰基)-L-Asp（PALA；双底物类似物）和 2 种阴离子抑制剂（Cl− 和 ATP）的情况下，研究了尿素诱导的大肠杆菌天冬氨酸转氨甲酰酶催化三聚体 (C3) 低温解离和解折叠的途径和能量学分析和紫外差示光谱。在没有活性位点配体且存在 ATP 的情况下，c3 将低于 2 M 的尿素解离成膨胀的 c 链，然后逐渐从 2 M 尿素展开到 6 M 尿素，几乎没有明显的协同作用。通过 3 种独立类型的实验确定的自由能线性外推至 0 M 尿素，得出每个界面在 7.5 °C 下约 7–10 kcal m−1 的 ΔG 解离估计值。当建模为二态过程时，解离链的 ΔGunfolding 估计非常小，大约为 2 kcal m−1。这些数据也与解离单体的展开是 1 态膨胀过程的可能性一致。在存在配体 CP 和 PALA 的情况下，以及在存在 Cl− 的情况下，c3 在更高的尿素浓度下解离，并且三聚体解离和解折叠同时发生并且明显协同发生，尿素浓度随着配体的亲和力而增加。
We have examined the pathway and energetics of urea‐induced dissociation and unfolding of the catalytic trimer (C3) of aspartate transcarbamylase fromEscherichia coliat low temperature in the absence and presence of carbamyl phosphate (CP; a substrate),N‐(phosphonacetyl)‐L‐Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl−and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy. In the absence of active‐site ligands and in the presence of ATP, c3dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity. Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for ΔGdissociationat 7.5 °C of about 7–10 kcal m−1per interface. ΔGunfoldingof dissociated chains when modeled as a 2‐state process is estimated to be very small, on the order of ˜2 kcal m−1. The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1‐state swelling process. In the presence of the ligands CP and PALA, and in the presence of Cl−, c3dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand.