Braxton B L, Mullins L S, Raushel F M, Reinhart G D
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019-0370.
Biochemistry. 1992 Mar 3;31(8):2309-16. doi: 10.1021/bi00123a015.
The effects of the allosteric ligands UMP, IMP, and ornithine on the partial reactions catalyzed by Escherichia coli carbamyl phosphate synthetase have been examined. Both of these reactions, a HCO3(-)-dependent ATP synthesis reaction and a carbamyl phosphate-dependent ATP synthesis reaction, follow bimolecular ordered sequential kinetic mechanisms. In the ATPase reaction, MgATP binds before HCO3- as established previously for the overall reaction catalyzed by carbamyl phosphate synthetase [Raushel, F. M., Anderson, P. M., & Villafranca, J. J. (1978) Biochemistry 17, 5587-5591]. The initial velocity kinetics for the ATP synthesis reaction indicate that MgADP binds before carbamyl phosphate in an equilibrium ordered mechanism except in the presence of ornithine. Determination of true thermodynamic linked-function parameters describing the impact of allosteric ligands on the binding interactions of the first substrate to bind in an ordered mechanism requires experiments to be performed in which both substrates are varied even if only one is apparently affected by the allosteric ligands. In so doing, we have found that IMP has little effect on the overall reaction of either of these two partial reactions. UMP and ornithine, which have a pronounced effect on the apparent Km for MgATP in the overall reaction, both substantially change the thermodynamic dissociation constant for MgADP from the binary E-MgADP complex, Kia, in the ATP synthesis reaction, with UMP increasing Kia 15-fold and ornithine decreasing Kia by 18-fold. By contrast, only UMP substantially affects the Kia for MgATP in the ATPase reaction, increasing it by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
已研究了变构配体UMP、IMP和鸟氨酸对大肠杆菌氨甲酰磷酸合成酶催化的部分反应的影响。这两个反应，即依赖HCO3(-)的ATP合成反应和依赖氨甲酰磷酸的ATP合成反应，均遵循双分子有序序列动力学机制。在ATP酶反应中，MgATP在HCO3-之前结合，这与之前氨甲酰磷酸合成酶催化的整体反应情况一致[劳斯赫尔，F.M.，安德森，P.M.，& 维拉弗兰卡，J.J.（1978年）《生物化学》17，5587 - 5591]。ATP合成反应的初速度动力学表明，除了在鸟氨酸存在的情况下，MgADP在氨甲酰磷酸之前以平衡有序机制结合。确定描述变构配体对有序机制中首先结合的第一个底物的结合相互作用影响的真实热力学连锁函数参数，需要进行两个底物都变化的实验，即使只有一个底物明显受变构配体影响。通过这样做，我们发现IMP对这两个部分反应中的任何一个的整体反应影响很小。UMP和鸟氨酸在整体反应中对MgATP的表观Km有显著影响，它们在ATP合成反应中都极大地改变了MgADP从二元E - MgADP复合物的热力学解离常数Kia，UMP使Kia增加15倍，鸟氨酸使Kia降低18倍。相比之下，只有UMP在ATP酶反应中对MgATP的Kia有显著影响，使其增加5倍。（摘要截短于250字）
