Rochet J C, Brownie E R, Oikawa K, Hicks L D, Fraser M E, James M N, Kay C M, Bridger W A, Wolodko W T
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Biochemistry. 2000 Sep 19;39(37):11291-302. doi: 10.1021/bi0003184.
Pig heart CoA transferase (EC 2.8.3.5) has been shown previously to adopt a homodimeric structure, in which each subunit has a molecular weight of 52 197 and consists of N- and C-domains linked by a hydrophilic linker or "hinge". Here we identify and characterize a second oligomeric form constituent in purified enzyme preparations, albeit at low concentrations. Both species catalyze the transfer of CoA with similar values for k(cat) and K(M). This second form sediments more rapidly than the homodimer under the conditions of conventional sedimentation velocity and active enzyme centrifugation. Apparent molecular weight values determined by sedimentation equilibrium and gel filtration chromatography are 4-fold greater than the subunit molecular weight, confirming that this form is a homotetramer. The subunits of both oligomeric forms are indistinguishable with respect to molecular mass, far-UV CD, intrinsic tryptophan fluorescence, and equilibrium unfolding. Dissociation of the homotetramer to the homodimer occurs very slowly in benign solutions containing high salt concentrations (0.25-2.0 M KCl). The homotetramer is fully converted to homodimer during refolding from denaturant at low protein concentrations. Disruption of the hydrophilic linker between the N- and C-domains by mutagenesis or mild proteolysis causes a decrease in the relative amount of the larger conformer. The homotetramer is stabilized by interactions involving the helical hinge region, and a substantial kinetic barrier hinders interconversion of the two oligomeric species under nondenaturing conditions.
猪心脏辅酶A转移酶（EC 2.8.3.5）先前已被证明具有同二聚体结构，其中每个亚基的分子量为52197，由通过亲水性连接子或“铰链”连接的N结构域和C结构域组成。在这里，我们鉴定并表征了纯化酶制剂中的第二种寡聚体形式成分，尽管其浓度较低。两种形式催化辅酶A转移的k(cat)和K(M)值相似。在传统沉降速度和活性酶离心条件下，这种第二种形式的沉降速度比同二聚体更快。通过沉降平衡和凝胶过滤色谱法测定的表观分子量值比亚基分子量大四倍，证实这种形式是同四聚体。两种寡聚体形式的亚基在分子量、远紫外圆二色性、内在色氨酸荧光和平衡去折叠方面无法区分。在含有高盐浓度（0.25 - 2.0 M KCl）的良性溶液中，同四聚体解离为同二聚体的过程非常缓慢。在低蛋白浓度下从变性剂中重折叠时，同四聚体完全转化为同二聚体。通过诱变或温和的蛋白酶解破坏N结构域和C结构域之间的亲水性连接子会导致较大构象体的相对量减少。同四聚体通过涉及螺旋铰链区的相互作用得以稳定，并且在非变性条件下，一个实质性的动力学屏障阻碍了两种寡聚体形式的相互转化。
