Keruchenko J S, Keruchenko I D, Gladilin K L, Zaitsev V N, Chirgadze N Y
A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow.
Biochim Biophys Acta. 1992 Jul 13;1122(1):85-92. doi: 10.1016/0167-4838(92)90131-v.
Fumarase (fumarate hydratase, EC 4.2.1.2) from Saccharomyces cerevisiae has been purified to homogeneity by a method including acetone fractionation, DEAE ion-exchange and dye-sorbent affinity chromatography. The suggested method allows fumarase purification with a yield higher than 60% and may be used to obtain large enzyme quantities. The native protein consists of four subunits with a approximately 50 kDa molecular mass each and has an isoelectric point at pH 6.5 +/- 0.3. The equilibrium constant for fumarate hydration is about 4.3 (25 degrees C, pH 7.5), the Michaelis constants for fumarate and 1-malate are approximately 30 microM and approximately 250 microM, respectively. The enzyme is activated by substrates and multivalent anions, the activation seems to be of a non-competitive type. The fumarase complex with meso-tartaric acid has been crystallized by the vapor diffusion method. The unit cell parameters are a = 93.30, b = 94.05 and c = 106.07 A, space group P2(1)2(1)2(1). The unit cell contains 2 protein molecules. The crystals diffract to at least 2.6 A resolution and are suitable for X-ray structure analysis.
通过包括丙酮分级分离、DEAE离子交换和染料吸附亲和色谱在内的方法，已将来自酿酒酵母的延胡索酸酶（延胡索酸水合酶，EC 4.2.1.2）纯化至同质。所建议的方法可实现产率高于60%的延胡索酸酶纯化，可用于获得大量该酶。天然蛋白质由四个亚基组成，每个亚基的分子量约为50 kDa，其等电点为pH 6.5±0.3。延胡索酸水合的平衡常数约为4.3（25℃，pH 7.5），延胡索酸和L-苹果酸的米氏常数分别约为30 μM和约250 μM。该酶被底物和多价阴离子激活，这种激活似乎是非竞争性的。与内消旋酒石酸形成的延胡索酸酶复合物已通过气相扩散法结晶。晶胞参数为a = 93.30、b = 94.05和c = 106.07 Å，空间群为P2(1)2(1)2(1)。每个晶胞包含2个蛋白质分子。这些晶体的衍射分辨率至少为2.6 Å，适合进行X射线结构分析。
