Kamemoto E S, Iltzsch M H, Lan L, Mansour T E
Department of Pharmacology, Stanford University School of Medicine, California 94305.
Arch Biochem Biophys. 1987 Oct;258(1):101-11. doi: 10.1016/0003-9861(87)90327-4.
Phosphofructokinase from the liver fluke, Fasciola hepatica, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase isolated from this organism. Phosphorylated fluke phosphofructokinase had a sevenfold lower apparent Km for its substrate, Fru-6-P, and an eightfold higher 0.5 Vopt for ATP, the enzyme's primary inhibitor, than native phosphofructokinase. Activation of fluke phosphofructokinase following phorphorylation by a mammalian protein kinase catalytic subunit was previously reported (E. S. Kamemoto and T. E. Mansour (1986) J. Biol. Chem. 261, 4346-4351). The catalytic subunit of protein kinase isolated from the liver fluke phosphorylated sites on fluke phosphofructokinase similar to those phosphorylated by the mammalian enzyme. Maximal phosphate incorporation was 0.3 mol P/mol of protomer. The native enzyme was found to contain 1.3 mol P/mol of protomer. In contrast to fluke phosphofructokinase, activity of the mammalian heart enzyme was slightly decreased following phosphorylation. The dependence of allosteric interaction on an acidic pH observed with the mammalian phosphofructokinase was not observed with the fluke enzyme. Unlike mammalian phosphofructokinase, allosteric kinetics of the fluke enzyme was observed at alkaline pH (8.0). Fluke phosphofructokinase was found to be relatively insensitive to inhibition by citrate, a known potent inhibitor of the mammalian enzyme. Fru-2,6-P2, a potent modifier of phosphofructokinase from a variety of sources, was found to activate both native and phosphorylated fluke phosphofructokinase. The most potent activators of fluke phosphofructokinase were found to be Fru-2,6-P2, AMP, and phosphorylation. The endogenous level of Fru-2,6-P2 in the flukes was determined to be 29 +/- 1.3 nmol/g wet wt, a level that may well modulate enzyme activity. Fru-6-P,2-kinase, the enzyme responsible for synthesis of Fru-2,6-P2, was found to be present in the flukes. Our results suggest physiological roles for phosphorylation and Fru-2,6-P2 in regulation of fluke phosphofructokinase.
来自肝吸虫（肝片吸虫）的磷酸果糖激酶被从该生物体中分离出的环磷酸腺苷依赖性蛋白激酶的催化亚基磷酸化。与天然磷酸果糖激酶相比，磷酸化的吸虫磷酸果糖激酶对其底物果糖-6-磷酸（Fru-6-P）的表观Km值低7倍，对该酶的主要抑制剂ATP的0.5Vopt高8倍。先前有报道称，哺乳动物蛋白激酶催化亚基磷酸化后，吸虫磷酸果糖激酶会被激活（E.S. Kamemoto和T.E. Mansour（1986年）《生物化学杂志》261卷，4346 - 4351页）。从肝吸虫中分离出的蛋白激酶催化亚基使吸虫磷酸果糖激酶上的磷酸化位点与哺乳动物酶磷酸化的位点相似。最大磷掺入量为0.3摩尔磷/摩尔原体。发现天然酶含有1.3摩尔磷/摩尔原体。与吸虫磷酸果糖激酶不同，哺乳动物心脏酶磷酸化后活性略有下降。在哺乳动物磷酸果糖激酶中观察到的变构相互作用对酸性pH的依赖性在吸虫酶中未观察到。与哺乳动物磷酸果糖激酶不同，吸虫酶在碱性pH（8.0）下观察到变构动力学。发现吸虫磷酸果糖激酶对柠檬酸盐（一种已知的哺乳动物酶的有效抑制剂）的抑制相对不敏感。果糖-2,6-二磷酸（Fru-2,6-P2）是来自多种来源的磷酸果糖激酶的有效调节剂，被发现可激活天然和磷酸化的吸虫磷酸果糖激酶。发现吸虫磷酸果糖激酶最有效的激活剂是Fru-2,6-P2、AMP和磷酸化。测定吸虫中Fru-2,6-P2的内源性水平为29±1.3纳摩尔/克湿重，这一水平很可能调节酶的活性。发现负责合成Fru-2,6-P2的果糖-6-磷酸,2-激酶存在于吸虫中。我们的结果表明磷酸化和Fru-2,6-P2在调节吸虫磷酸果糖激酶中具有生理作用。
