Sprenger G A, Schörken U, Sprenger G, Sahm H
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Germany.
Eur J Biochem. 1995 Jun 1;230(2):525-32. doi: 10.1111/j.1432-1033.1995.0525h.x.
Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19-derived plasmid. These recombinant cells exhibited a transketolase activity in crude extracts of up to 9.7 U/mg compared to < or = 0.1 U/mg in wild-type cells. Transketolase A was purified from crude extracts of a recombinant strain by successive ammonium sulfate precipitations and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE column) and afforded an apparently homogeneous protein band on SDS/PAGE. The enzyme, both in its active and apoform, had a molecular mass of 145,000 Da (+/- 10,000 Da), judged by gel-filtration chromatography. Subunits of 73,000 Da (+/- 2000 Da) were determined on SDS/PAGE, thus, transketolase A most likely forms a homodimer. N-terminal amino acid sequencing of the protein verified the identity with the cloned gene tktA. The specific activity of the purified enzyme, determined at 30 degrees C with the substrates xylulose 5-phosphate (donor of C2 compound) and ribose 5-phosphate (acceptor) at an optimal pH (50 mM glycylglycine, pH 8.5), was 50.4 U/mg. Km values for the substrates xylulose 5-phosphate and ribose 5-phosphate were 160 microM and 1.4 mM, respectively. Km values for the other physiological substrates of transketolase A were 90 microM for erythrose 4-phosphate (best acceptor substrate), 2.1 mM for D,L-glyceraldehyde 3-phosphate, 1.1 mM for fructose 6-phosphate, and 4 mM for sedoheptulose 7-phosphate. Hydroxypyruvate served as alternative donor (Km = 18 mM). Unphosphorylated acceptor compounds were formaldehyde (Km = 31 mM), glycolaldehyde (14 mM), D,L-glyceraldehyde (10 mM) and D-erythrose (150 mM). The enzyme was competitively inhibited by D-arabinose 5-phosphate (K = 6 mM at a concentration of 2.5 mM D-arabinose 5-phosphate) or by the chelating agent EDTA. The inactive apoform of transketolase A was yielded by dialysis against buffer containing 10 mM EDTA, thus removing the cofactors thiamine diphosphate and divalent cations. The reconstitution of the apoenzyme proceded faster in the presence of manganese ions (Kd = 7 microM at 10 microM thiamine diphosphate) than with other divalent cations.
转酮醇酶A是从携带同源克隆的tktA基因的重组大肠杆菌K12细胞中纯化至表观均一性的，该基因位于pUC19衍生质粒上。与野生型细胞中≤0.1 U/mg相比，这些重组细胞粗提物中的转酮醇酶活性高达9.7 U/mg。通过连续的硫酸铵沉淀和两步阴离子交换色谱法（Q-Sepharose FF、Fractogel EMD-DEAE柱）从重组菌株的粗提物中纯化转酮醇酶A，并在SDS/PAGE上得到一条表观均一的蛋白条带。通过凝胶过滤色谱法判断，该酶的活性形式和脱辅基形式的分子量均为145,000 Da（±10,000 Da）。在SDS/PAGE上测定的亚基分子量为73,000 Da（±2000 Da），因此，转酮醇酶A很可能形成同型二聚体。该蛋白的N端氨基酸测序证实了其与克隆基因tktA的一致性。在30℃下，以5-磷酸木酮糖（C2化合物供体）和5-磷酸核糖（受体）为底物，在最佳pH值（50 mM甘氨酰甘氨酸，pH 8.5）下测定的纯化酶的比活性为50.4 U/mg。5-磷酸木酮糖和5-磷酸核糖底物的Km值分别为160 μM和1.4 mM。转酮醇酶A的其他生理底物的Km值分别为：4-磷酸赤藓糖为90 μM（最佳受体底物）、3-磷酸甘油醛为2.1 mM、6-磷酸果糖为1.1 mM、7-磷酸景天庚酮糖为4 mM。羟基丙酮酸作为替代供体（Km = 18 mM）。未磷酸化的受体化合物为甲醛（Km = 31 mM）、乙醇醛（14 mM）、3-磷酸甘油醛（10 mM）和D-赤藓糖（150 mM）。该酶被5-磷酸D-阿拉伯糖（在2.5 mM 5-磷酸D-阿拉伯糖浓度下K = 6 mM）或螯合剂EDTA竞争性抑制。通过用含有10 mM EDTA的缓冲液透析产生转酮醇酶A的无活性脱辅基形式，从而去除辅因子硫胺素二磷酸和二价阳离子。在锰离子存在下（在10 μM硫胺素二磷酸时Kd = 7 μM），脱辅基酶的重构比其他二价阳离子存在时更快。
