Braxton B L, Mullins L S, Raushel F M, Reinhart G D
Department of Chemistry, University of Wisconsin-La Crosse 54601, USA.
Biochemistry. 1996 Sep 10;35(36):11918-24. doi: 10.1021/bi961305m.
When catalyzing the formation of MgATP and carbamate from MgADP and carbamoyl phosphate, Escherichia coli carbamoyl phosphate synthetase (CPS) binds MgADP with a large negative change in heat capacity. The magnitude of this heat capacity change is not appreciably altered by the presence of a saturating concentration of either the allosteric activator ornithine or the inhibitor UMP despite the substantial and opposing effects these ligands have on the binding affinity for MgADP. By contrast, no detectable change in heat capacity is associated with the thermodynamic coupling between MgADP and either ornithine or UMP. The sign of the apparently constant enthalpic and entropic contributions to the coupling free energy for each of these ligands is opposite that of the coupling free energy, indicating that the observed allosteric phenomenology is in net opposed by the enthalpy of the interaction and instead arises from a change in entropy of the system. IMP produces only a very small allosteric effect as indicated by a near-zero value for the MgADP-IMP coupling free energy. However, the enthalpic and entropic contributions are individually larger in absolute value for the IMP coupling than for those pertaining to the other allosteric ligands, and entropy dominates the coupling free energy above 36 degrees C, causing IMP to become an activator at high temperature. In addition, the sign of the coupling enthalpy and entropy for IMP has the same sign as the coupling enthalpy and entropy produced by ornithine, suggesting that IMP and ornithine may similarly influence the enzyme at a molecular level despite binding to different allosteric sites on the enzyme. The data are consistent with a model in which the actions of the allosteric ligands arise primarily from changes in the conformational degeneracy introduced by each ligand. With this model, one can also rationalize the failure of these allosteric ligands to substantially influence kcat.
在催化由MgADP和氨甲酰磷酸形成MgATP和氨基甲酸盐的过程中，大肠杆菌氨甲酰磷酸合成酶（CPS）结合MgADP时伴随着较大的负热容变化。尽管变构激活剂鸟氨酸或抑制剂UMP的饱和浓度对MgADP的结合亲和力有显著且相反的影响，但这种热容变化的幅度并未因它们的存在而明显改变。相比之下，MgADP与鸟氨酸或UMP之间的热力学偶联没有可检测到的热容变化。这些配体中每个配体对偶联自由能的明显恒定的焓和熵贡献的符号与偶联自由能的符号相反，这表明观察到的变构现象在净效应上与相互作用的焓相反，而是源于系统熵的变化。IMP产生的变构效应非常小，MgADP-IMP偶联自由能接近零值就表明了这一点。然而，IMP偶联的焓和熵贡献在绝对值上分别比其他变构配体的更大，并且在36摄氏度以上熵主导偶联自由能，导致IMP在高温下成为激活剂。此外，IMP的偶联焓和熵的符号与鸟氨酸产生的偶联焓和熵的符号相同，这表明尽管IMP和鸟氨酸结合在酶的不同变构位点上，但它们可能在分子水平上对酶产生类似的影响。这些数据与一个模型一致，在该模型中变构配体的作用主要源于每个配体引入的构象简并度的变化。基于这个模型，也可以解释这些变构配体未能显著影响kcat的原因。
