Rubio V, Britton H G, Grisolia S
Eur J Biochem. 1979 Jan 15;93(2):245-56. doi: 10.1111/j.1432-1033.1979.tb12817.x.
This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate. This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of [gamma-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321--329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.
本文通过脉冲追踪技术证明，将其γ-磷酸基团转移至氨甲酰磷酸的ATP分子（ATPB）可与大鼠肝脏线粒体氨甲酰磷酸合成酶结合。这种结合的ATPB在与游离ATP交换之前，可与NH₃、HCO₃⁻和ATP（见下文）反应生成氨甲酰磷酸。这种结合需要Mg²⁺和N-乙酰谷氨酸，但不需要NH₃或HCO₃⁻；结合的量取决于ATP的浓度（Kapp = 10 - 30 μM ATP）和酶的量。在饱和状态下，每个酶二聚体至少结合一个ATPB分子。ATPB的结合遵循缓慢的指数时间进程（t1/2为8 - 16秒，22℃），与ATP浓度无关，且几乎不受NH₃、NCO₃⁻或在[γ-³²P]ATP脉冲之前用未标记的ATP孵育酶的影响。当酶与ATP一起孵育时，由微量的NH₃和HCO₃⁻形成氨甲酰磷酸遵循从结合的ATPB生成时预期的动力学，表明后者是正常酶反应中氨甲酰磷酸（“Cbm-P前体”）的前体。这表明ATPB的位点在溶液中通常对ATP不可及，但当酶经历周期性构象变化时变得可及。当酶恢复到不可及的构象时，结合的ATP成为Cbm-P前体。在没有NH₃和HCO₃⁻（小于0.2 mM）的情况下进行的脉冲追踪实验也证明了ATPA（在正常酶反应中产生Pi的分子）的结合，如³²Pi产生中的“爆发”所示。因此，（根据我们之前的发现）ATPA和ATPB都可以同时与酶结合，并在与游离ATP交换之前在追踪溶液中与NH₃和HCO₃⁻反应。然而，在脉冲孵育中ATP浓度较低（18 μM）时，只有ATPB结合，因为追踪中需要ATP（见上文）。尽管存在两个ATP结合位点，但双功能抑制剂腺苷（5'）五磷酸（5'）腺苷（Ap5A）未能显著抑制该酶。提出了对先前发表的方案[Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321 - 329]的更详细修改；建议当产物离开酶且活性中心处于可及构型时，ATPB可进入活性中心。从可及构型到不可及构型的转变似乎是正常酶反应的一部分，并且可能代表其他人从稳态动力学推测的构象变化。中间体的性质还表明，ATPA的水解必须在很大程度上负责该酶的HCO₃⁻依赖性ATP酶活性。Ap5A对该酶缺乏抑制作用表明大肠杆菌和大鼠肝脏合成酶之间存在实质性差异。
