Mort J S, Sanwal B D
Can J Biochem. 1978 Jun;56(6):647-53. doi: 10.1139/o78-097.
The pyruvate kinase activated by ribose-5-phosphate from Escherichia coli has been purified to homogeneity, taking advantage of the stabilization of the enzyme by its inhibitor phosphate and by thiol reagents. The native enzyme has a tetrameric quaternary structure which, while prone to dissociation under many conditions, remains intact in the presence of the above reagents. The enzyme was found to reactivate on dilution out of 8 M urea. Interestingly, the recovery of activity is greatly increased by phosphate, an allosteric inhibitor, but markedly reduced by the allosteric activator, ribose-5-phosphate, implying that it is harder for the enzyme to refold to a 'relaxed state.' Proteolysis studies indicate a more open structure in the presence of the activator.
利用磷酸及其抑制剂和硫醇试剂对来自大肠杆菌的由5-磷酸核糖激活的丙酮酸激酶进行了纯化，直至达到同质状态。天然酶具有四聚体四级结构，虽然在许多条件下易于解离，但在上述试剂存在下仍保持完整。发现该酶在从8M尿素中稀释出来后会重新激活。有趣的是，变构抑制剂磷酸可大大提高活性恢复率，但变构激活剂5-磷酸核糖则会使其明显降低，这意味着该酶更难重新折叠成“松弛状态”。蛋白水解研究表明，在激活剂存在下结构更为开放。
