Bergström G, Ekman P, Humble E, Engström L
Biochim Biophys Acta. 1978 Feb 15;532(2):259-67. doi: 10.1016/0005-2795(78)90580-9.
The phosphorylated or phosphate-accepting site of pyruvate kinase from pig and rat liver was removed without inactivation by incubation with subtilisin. At different time intervals the subtilisin was inactivated with phenylmethylsulfonyl fluoride and the amount of remaining phosphorylatable or phosphorylated sites of pyruvate kinase estimated by incubation with an excess of [32P]-ATP and protein kinase. It was found that to get the same rate of modification the subtilisin concentration required to modify unphosphorylated pyruvate kinase was approximately ten times higher than that used for removal of the phosphorylated site of phosphorylated site of phosphorylated enzyme. It was shown that the proteolytically-modified pyruvate kinase had an increased apparent Km for phosphoenolpyruvate without a change in V, when compared to unmodified unphosphorylated and phosphorylated pyruvate kinase. The removal of the phosphorylated site was not associated with loss of the allosteric sites for ATP and Fru-1,6-P2. The possibility that phosphorylation of the pyruvate kinase increases its degradation rate in vivo is briefly discussed.
猪和大鼠肝脏丙酮酸激酶的磷酸化位点或磷酸接受位点在与枯草杆菌蛋白酶孵育时被去除，且酶未失活。在不同时间间隔，用苯甲基磺酰氟使枯草杆菌蛋白酶失活，并通过与过量的[32P] -ATP和蛋白激酶孵育来估计丙酮酸激酶剩余的可磷酸化或已磷酸化位点的数量。结果发现，为了获得相同的修饰速率，修饰未磷酸化丙酮酸激酶所需的枯草杆菌蛋白酶浓度大约是用于去除磷酸化酶磷酸化位点的浓度的十倍。结果表明，与未修饰的未磷酸化和磷酸化的丙酮酸激酶相比，经蛋白水解修饰的丙酮酸激酶对磷酸烯醇丙酮酸的表观Km增加，而V不变。磷酸化位点的去除与ATP和Fru-1,6-P2变构位点的丧失无关。文中简要讨论了丙酮酸激酶磷酸化在体内增加其降解速率的可能性。
