REVERSIBLE DISSOCIATION AND UNFOLDING OF ASPARTATE-AMINOTRANSFERASE FROM ESCHERICHIA-COLI - CHARACTERIZATION OF A MONOMERIC INTERMEDIATE
REVERSIBLE DISSOCIATION AND UNFOLDING OF ASPARTATE-AMINOTRANSFERASE FROM ESCHERICHIA-COLI - CHARACTERIZATION OF A MONOMERIC INTERMEDIATE
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DOI：
10.1021/bi00459a035
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发表时间：
1990-02-20
期刊：
影响因子：
2.9
通讯作者：
KIRSCHNER, K
中科院分区：
文献类型：
--
作者：
HEROLD, M;KIRSCHNER, K
研究了盐酸胍对大肠杆菌天冬氨酸氨基转移酶（D）二聚体的解折叠和解离。根据0.7 mg变性蛋白/mL对缓冲液透析后酶活性几乎完全恢复判断，整个过程是可逆的。展开和解离进行监测，圆二色性和荧光光谱，并发生在三个单独的阶段：D. dblarw。2M. dblarw. 2M*. dblarw. 2U。在约0.5 M盐酸胍时的第一次转变与酶活性的丧失相一致。通过吡哆醛5“-磷酸盐或吡哆胺5”-磷酸盐的存在使其向更高的变性剂浓度位移，并且通过降低蛋白质浓度使其向更低的变性剂浓度位移。因此，结合的辅酶稳定二聚体状态，并且单体（M）是无活性的，因为共享的活性位点被二聚体的解离破坏。M转化为M*，然后在两个随后的转变中转化为完全未折叠的单体（U）。M* 在0.9和1.1 M盐酸胍之间是稳定的，并且具有单体的、紧凑的“熔融球”状态的流体动力学半径、圆二色性和荧光。
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 .dblarw. 2M .dblarw. 2M* .dblarw. 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 dissociaton 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.