Newell J O, Markby D W, Schachman H K
Department of Molecular Biology, University of California, Berkeley 94720.
J Biol Chem. 1989 Feb 15;264(5):2476-81.
Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The isolated catalytic subunit of ATCase, which lacks the cooperative kinetic properties of the holoenzyme, exhibits only a very slight degree of cooperativity in binding PALA. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation. Ligation of the regulatory subunits by either of the allosteric effectors leads to a change in the effect of PALA on the association-dissociation equilibrium.
大多数关于大肠杆菌中调节酶天冬氨酸转氨甲酰酶（ATCase）变构性质的研究都是基于酶活性对底物浓度的S形依赖性，以及抑制剂CTP和激活剂ATP对饱和曲线的影响。由于无法区分变构转变过程中底物结合和催化周转的变化，因此用分子模型来解释这些效应变得很复杂。为了消除这种模糊性，通过在pH 7的磷酸盐缓冲液中进行平衡透析，直接测量了3H标记的双底物类似物N-（膦酰基乙酰基）-L-天冬氨酸（PALA）在不存在和存在变构效应物ATP和CTP的情况下与天冬氨酸转氨甲酰酶的结合。PALA以显著的协同性与全酶结合，平均解离常数为110 nM。ATP和CTP以类似于它们对酶动力学性质的影响方式，改变了ATCase对PALA的平均亲和力以及结合过程中的协同程度；在ATP存在下，PALA的平均解离常数降至65 nM，在CTP存在下增加至266 nM，而在不存在效应物时Hill系数为1.95，在ATP和CTP存在下分别变为1.35和2.27。缺乏全酶协同动力学性质的ATCase分离催化亚基在结合PALA时仅表现出非常轻微的协同程度。PALA与催化亚基的解离常数为95 nM。根据将PALA结合与由催化亚基和调节亚基组装ATCase联系起来的热力学方案对这些结果进行解释表明，用PALA使酶饱和会使全酶和亚基之间的平衡略微向解离方向移动。变构效应物中的任何一种对调节亚基的连接都会导致PALA对缔合-解离平衡的影响发生变化。
