Communication between dissimilar subunits in aspartate transcarbamoylase: effect of inhibitor and activator on the conformation of the catalytic polypeptide chains.
Communication between dissimilar subunits in aspartate transcarbamoylase: effect of inhibitor and activator on the conformation of the catalytic polypeptide chains.
复制标题
天冬氨酸转氨甲酰酶中不同亚基之间的通讯：抑制剂和激活剂对催化多肽链构象的影响。
DOI：
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发表时间：
1979
影响因子：
11.1
通讯作者：
H. K. Schachman
中科院分区：
文献类型：
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作者：
P. Hensley;H. K. Schachman
尽管配体与蛋白质结合的局部直接效应在概念上容易与远离结合位点的蛋白质区域的总体间接构象变化区分开来，但在实验上很难区分它们。例如，在寡聚蛋白质中，配体与一条链的结合可能导致无配体链的构象变化，但许多物理化学探针没有足够的区分力来证明变化发生的位置。关于抑制剂CTP或激活剂ATP在与变构酶天冬氨酸氨甲酰转移酶（天冬氨酸氨甲酰转移酶;氨甲酰磷酸：L-天冬氨酸氨甲酰转移酶，EC 2.1.3.2）的调节链结合时是否引起构象变化，该构象变化在整个酶中传播到催化链，一直缺乏证据。为了证明这种“通信”的影响，在一个网站上的其他多肽链的构象结合，我们构建了分子含有天然的调节亚基和酶活性硝化催化亚基具有一个敏感的硝基酪氨酸生色团每条多肽链。这些杂合分子表现出天然酶的特征性调节特性。在添加CTP后，分子群体向约束或T状态转移，如沉降系数的变化和酶动力学的改变所示。此外，由于硝化催化多肽链的环境改变，在430 nm处的吸光度降低。与此相反，ATP引起的位移向放松或R构象，和吸光度由于硝基酪氨酸残基增加。不同类型的实验表明，改性的酶分子是在一个预先存在的平衡，是由CTP或ATP的扰动，硝化催化亚基中产生的构象变化检测到相反的改变，在其吸收光谱。
Although local, direct effects of ligand binding to proteins are readily differentiated conceptually from gross, indirect conformational changes in regions of the protein remote from the site of binding, it has been difficult experimentally to distinguish between them. In oligomeric proteins, for example, the binding of ligands to one chain may cause a conformational change in the unliganded chains, but many physical chemical probes are not sufficiently discriminating to demonstrate where the change occurred. Evidence has been lacking as to whether the inhibitor, CTP, or the activator, ATP, in binding to the regulatory chains of the allosteric enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) causes a conformational change that is propagated throughout the enzyme to the catalytic chains. To demonstrate this "communication" of effects of binding at one site on the conformation of other polypeptide chains, we constructed molecules containing native regulatory subunits and enzymically active nitrated catalytic subunits having one sensitive nitrotyrosyl chromophore per polypeptide chain. These hybrid molecules exhibited the characteristic regulatory properties of the native enzyme. Upon the addition of CTP the population of molecules was shifted toward the constrained or T state, as shown by the change in the sedimentation coefficient and the altered enzyme kinetics. Moreover, there was a decrease in absorbance at 430 nm due to the altered environment of the nitrated catalytic polypeptide chains. In contrast, ATP caused a shift toward the relaxed or R conformation, and the absorbance due to the nitrotyrosyl residues was increased. Different types of experiments indicated that the modified enzyme molecules are in a preexisting equilibrium that is perturbed by CTP or ATP; the resulting conformational changes in the nitrated catalytic subunits are detected by opposite alterations in their absorbance spectrum.