Herold M, Leistler B, Hage A, Luger K, Kirschner K
Abteilung für Biophysikalische Chemie, Universität Basel, Switzerland.
Biochemistry. 1991 Apr 16;30(15):3612-20. doi: 10.1021/bi00229a004.
The coenzyme (PLP) binding domain (residues 47-329) of the dimeric aspartate aminotransferase from Escherichia coli was produced separately by recombinant DNA methods. It folded autonomously both in vivo and in vitro, that is, independently of the native N- and C-terminal extensions that combine to form the small domain of eAAT. The PLP-domain had one binding site for PLP of relatively high affinity involving a covalent bond to the protein. It was monomeric, although the major subunit-subunit interface at the 2-fold symmetry axis remained unchanged. This effect appears to be due mainly to the absence of the N-terminal extension that contains hydrophobic residues, which interact with the PLP-domain of the second subunit in the wild-type dimer. Judged by circular dichroism, fluorescence, and HPLC gel filtration at increasing concentrations of guanidinium chloride, the PLP-domain underwent a three-state unfolding transition (M' in equilibrium M'* in equilibrium U') involving a compact intermediate M'*. This behavior parallels the unfolding of the dissociated native monomer of cAAT.
通过重组DNA方法分别制备了来自大肠杆菌的二聚天冬氨酸转氨酶的辅酶（PLP）结合结构域（第47至329位氨基酸残基）。它在体内和体外均能自主折叠，也就是说，独立于天然的N端和C端延伸部分，这两个延伸部分结合形成了eAAT的小结构域。PLP结构域有一个与PLP亲和力相对较高的结合位点，涉及与蛋白质的共价键。它是单体形式，尽管在2倍对称轴处的主要亚基-亚基界面保持不变。这种效应似乎主要是由于缺少包含疏水残基的N端延伸部分，在野生型二聚体中，该延伸部分与第二个亚基的PLP结构域相互作用。通过圆二色性、荧光以及在逐渐增加的氯化胍浓度下进行的高效液相色谱凝胶过滤判断，PLP结构域经历了一个三态解折叠转变（M'处于平衡态M'*，M'处于平衡态U'），涉及一个紧密的中间体M'。这种行为与解离的天然cAAT单体的解折叠相似。
