El-Maghrabi M R, Pate T M, Murray K J, Pilkis S J
J Biol Chem. 1984 Nov 10;259(21):13096-103.
Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase catalyzes exchange reactions between ADP and ATP and between fructose-6-P and fructose-2,6-P2 at histidyl residues. Limited proteolysis of the enzyme with thermolysin yielded an enzyme core with a subunit molecular weight of 35,000-38,000. This enzyme core had no kinase activity and a 2-fold activated bisphosphatase activity whose sensitivity to the product inhibitor fructose-6-P was unchanged. The thermolysin-treated enzyme also did not catalyze the fructose-6-P/fructose-2,6-P2 exchange reaction but did catalyze the ADP/ATP exchange. These results suggest that 1) the enzyme's reactions may be catalyzed at two active sites, 2) there are at least two fructose-6-P binding sites, 3) the fructose-6-P/fructose-2,6-P2 exchange is catalyzed only at the kinase site, and 4) inactivation of the exchange and kinase reactions by thermolysin digestion is due to the loss of the fructose-6-P binding site of the kinase. Also consistent with these conclusions was the finding that oxidation of the enzyme with ascorbate/Fe3+ or H2O2 resulted in complete loss of the kinase activity as well as the fructose-6-P/fructose-2,6-P2 exchange but did not affect the bisphosphatase activity or the ADP/ATP exchange. Dithiothreitol could completely reactivate the ascorbate/Fe3+-inactivated enzyme, suggesting that oxidation occurred at a sulfhydryl group(s) essential for fructose-6-P binding in the kinase reaction. In addition, the kinase and fructose-6-P/fructose-2,6-P2 exchange reactions were more sensitive to inactivation by diethylpyrocarbonate than was the bisphosphatase. The different responses of the kinase and bisphosphatase reactions to the action of these various protein-modifying agents and to thermolysin digestion support the existence of a separate site for each reaction and an essential role for sulfhydryl groups at the sugar-phosphate-binding site(s) of the kinase.
大鼠肝脏6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶催化组氨酸残基处ADP与ATP之间以及果糖-6-磷酸与果糖-2,6-二磷酸之间的交换反应。用嗜热菌蛋白酶对该酶进行有限的蛋白水解，得到一个亚基分子量为35,000 - 38,000的酶核心。该酶核心没有激酶活性，但双磷酸酶活性提高了2倍，其对产物抑制剂果糖-6-磷酸的敏感性未改变。经嗜热菌蛋白酶处理的酶也不催化果糖-6-磷酸/果糖-2,6-二磷酸交换反应，但能催化ADP/ATP交换反应。这些结果表明：1）该酶的反应可能在两个活性位点催化；2）至少有两个果糖-6-磷酸结合位点；3）果糖-6-磷酸/果糖-2,6-二磷酸交换仅在激酶位点催化；4）嗜热菌蛋白酶消化导致交换反应和激酶反应失活是由于激酶的果糖-6-磷酸结合位点丧失。与这些结论一致的还有以下发现：用抗坏血酸盐/Fe3+或H2O2氧化该酶会导致激酶活性以及果糖-6-磷酸/果糖-2,6-二磷酸交换完全丧失，但不影响双磷酸酶活性或ADP/ATP交换。二硫苏糖醇能使抗坏血酸盐/Fe3+失活的酶完全重新激活，这表明氧化发生在激酶反应中果糖-6-磷酸结合所必需的一个或多个巯基上。此外，激酶和果糖-6-磷酸/果糖-2,6-二磷酸交换反应比双磷酸酶对焦碳酸二乙酯失活更敏感。激酶和双磷酸酶反应对这些各种蛋白质修饰剂的作用以及对嗜热菌蛋白酶消化的不同反应支持了每个反应存在一个独立位点以及激酶的糖磷酸结合位点处的巯基具有重要作用。
