Herold M, Kirschner K
Abteilung für Biophysikalische Chemie, Biozentrum, Universität Basel, Switzerland.
Biochemistry. 1990 Feb 20;29(7):1907-13. doi: 10.1021/bi00459a035.
The unfolding and dissociation of the dimeric enzyme aspartate aminotransferase (D) from Escherichia coli by guanidine hydrochloride have been investigated at equilibrium. The overall process was reversible, as judged from almost complete recovery of enzymic activity after dialysis of 0.7 mg of denatured protein/mL against buffer. Unfolding and dissociation were monitored by circular dichroism and fluorescence spectroscopy and occurred in three separate phases: D in equilibrium 2M in equilibrium 2M* in equilibrium 2U. The first transition at about 0.5 M guanidine hydrochloride coincided with loss of enzyme activity. It was displaced toward higher denaturant concentrations by the presence of either pyridoxal 5'-phosphate or pyridoxamine 5'-phosphate and toward lower denaturant concentrations by decreasing the protein concentration. Therefore, bound coenzyme stabilizes the dimeric state, and the monomer (M) is inactive because the shared active sites are destroyed by dissociation of the dimer. M was converted to M* and then to the fully unfolded monomer (U) in two subsequent transitions. M* was stable between 0.9 and 1.1 M guanidine hydrochloride and had the hydrodynamic radius, circular dichroism, and fluorescence of a monomeric, compact "molten globule" state.
在平衡条件下，研究了盐酸胍对来自大肠杆菌的二聚体酶天冬氨酸转氨酶（D）的展开和解离作用。从0.7mg/mL变性蛋白对缓冲液透析后酶活性几乎完全恢复可以判断，整个过程是可逆的。通过圆二色光谱和荧光光谱监测展开和解离过程，其发生在三个不同阶段：D处于平衡态→2M处于平衡态→2M处于平衡态→2U。在约0.5M盐酸胍时的第一次转变与酶活性丧失相吻合。在磷酸吡哆醛或磷酸吡哆胺存在时，它向更高变性剂浓度方向移动，而通过降低蛋白质浓度则向更低变性剂浓度方向移动。因此，结合的辅酶稳定二聚体状态，而单体（M）无活性，因为二聚体解离破坏了共享的活性位点。在随后的两个转变中，M转变为M，然后转变为完全展开的单体（U）。M*在0.9至1.1M盐酸胍之间稳定，具有单体紧密“熔球”状态的流体力学半径、圆二色性和荧光特性。
