HETEROTROPIC EFFECTORS PROMOTE A GLOBAL CONFORMATIONAL CHANGE IN ASPARTATE TRANSCARBAMOYLASE
HETEROTROPIC EFFECTORS PROMOTE A GLOBAL CONFORMATIONAL CHANGE IN ASPARTATE TRANSCARBAMOYLASE
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DOI：
10.1021/bi00467a019
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发表时间：
1990-04-17
期刊：
影响因子：
2.9
通讯作者：
SCHACHMAN, HK
中科院分区：
文献类型：
--
作者：
EISENSTEIN, E;MARKBY, DW;SCHACHMAN, HK
大肠杆菌的调节酶天冬氨酸氨甲酰转移酶（ATCase）所表现出的活性对底物浓度的S形依赖性通常归因于配体促进的酶的四级结构的变化。虽然一个全球性的构象变化，在ATCase后的配体结合的六个活性位点的一些是有据可查的，相应的改变后，添加抑制剂，CTP，或激活剂，ATP，野生型酶的结构尚未被检测到。这样的证据是必不可少的，以测试是否异向性，以及同向性，效果可以占定量的耦合平衡，涉及的酶的构象变化和优先结合的配体的一种构象或其他方面。这一证据现在已经用ATCase的突变形式获得，其中调节链中的Lys 143被Ala取代，从而扰乱酶中调节链和催化链之间界面处的相互作用，并使低活性、紧密（T）构象相对于高活性、溶胀（R）状态不稳定。差沉降速度的实验，包括测量的双底物类似物N-（膦酰基）-L-天冬氨酸的结合所引起的变化表明，突变酶的沉降系数之间的观察到的野生型ATCase的T和R状态。我们将结果解释为表明在pH7.0的磷酸盐缓冲液中的[T]/[R]比从约2 × 10 - 4降低到约2 × 10 - 4。101的野生型酶到2.7的r143 Ala ATCase。向突变酶中加入CTP导致[T]/[R]增加至34，而在ATP存在下，该比率降低至0.2。PALA结合的平衡透析测量表明，希尔系数，nH，这是1.4的情况下的效应，增加到2.0 CTP的存在下，并减少到1.0的加入ATP。根据Monod、Wyman和Changeux的双态模型对平衡结合数据进行分析，得到的[T]/[R]比与沉降速度实验推导的结果雅阁。这些结果提供了令人信服的证据，即异嗜性效应物引起ATCcase的平均四级结构的改变，从而通过扰乱T.dblarw.R平衡来调节酶的活性。此外，观察反驳的建议，只有局部的酶的结构发生变化后，CTP和ATP的结合。
The sigmoidal dependence of activity on substrate concentration exhibited by the regulatory enzyme aspartate transcarbamoylase (ATCase) of Escherichia coli is generally attributed to a ligand-promoted change in the quaternary structure of the enzyme. Although a global conformational change in ATCase upon the binding of ligands to some of the six active sites is well documented, a corresponding alteration in the structure of the wild-type enzyme upon the addition of the inhibitor, CTP, or the activator, ATP, has not been detected. Such evidence is essential for testing whether heterotropic, as well as homotropic, effects can be accounted for quantitatively in terms of coupled equilibria involving a conformational change in the enzyme and preferential binding of ligands to one conformation or the other. This evidence has now been obtained with a mutant form of ATCase in which Lys 143 in the regulatory chain was replaced by Ala, thereby perturbing interactions at the interface between the regulatory and catalytic chains in the enzyme and destabilizing the low-activity, compact (T) conformation relative to the high-activity, swollen (R) state. Difference sedimentation velocity experiments involving measurements of the changes caused by the binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate demonstrated that the sedimentation coefficient of the mutant enzyme was intermediate between that observed for the T and R states of wild-type ATCase. We interpret the results as indicating that the [T]/[R] ratio in phosphate buffer at pH 7.0 is reduced from about 2 .times. 101 for the wild-type enzyme to 2.7 for r143Ala ATCase. The addition of CTP to the mutant enzyme led to an increase in [T]/[R] to 34, whereas the ratio was lowered to 0.2 in the presence of ATP. Equilibrium dialysis measurements for the binding of PALA demonstrated that the Hill coefficient, nH, which was 1.4 in the absence of effectors, was increased to 2.0 in the presence of CTP and decreased to 1.0 by the addition of ATP. Analysis of the equilibrium binding data, in terms of the two-state model of Monod, Wyman, and Changeux, yielded [T]/[R] ratios in accord with those deduced from the sedimentation velocity experiments. These results provide convincing evidence that heterotropic effectors cause an alteration in the average quaternary structure of ATCcase and thereby regulate the activity of the enzyme by perturbing the T.dblarw.R equilibrium. Moreover, the observations refute suggestions that only local changes in the structure of the enzyme occur upon the binding of CTP and ATP.