Muniyappa K, Mendicino J
Mol Cell Biochem. 1984 Aug;63(1):21-32. doi: 10.1007/BF00230158.
The influence of fructose 2,6-bisphosphate on the activation of purified swine kidney phosphofructokinase as a function of the concentration of fructose 6P, ATP and citrate was investigated. The purified enzyme was nearly completely inhibited in the presence of 2 mM ATP. The addition of 20 nM fructose 2,6-P2 reversed the inhibition and restored more than 80% of the activity. In the absence of fructose 2,6-P2 the reaction showed a sigmoidal dependence on fructose-6-phosphate. The addition of 10 nM fructose 2,6-bisphosphate decreased the K0.5 for fructose 6-phosphate from 3 mM to 0.4 mM in the presence of 1.5 mM ATP. These results clearly show that fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate and decreases the inhibitory effect of ATP. The extent of inhibition by citrate was also significantly decreased in the presence of fructose 2,6-phosphate. The influence of various effectors of phosphofructokinase on the binding of ATP and fructose 6-P to the enzyme was examined in gel filtration studies. It was found that kidney phosphofructokinase binds 5.6 moles of fructose 6-P per mole of enzyme, which corresponds to about one site per subunit of tetrameric enzyme. The KD for fructose 6-P was 13 microM and in the presence of 0.5 mM ATP it increased to 27 microM. The addition of 0.3 mM citrate also increased the KD for fructose 6-P to about 40 microM. AMP, 10 microM, decreased the KD to 5 microM and the addition of fructose 2,6-phosphate decreased the KD for fructose 6-P to 0.9 microM. The addition of these compounds did not effect the maximal amount of fructose 6-P bound to the enzyme, which indicated that the binding site for these compounds might be near, but was not identical to the fructose 6-P binding site. The enzyme bound a maximum of about 12.5 moles of ATP per mole, which corresponds to 3 moles per subunit. The KD of the site with the highest affinity for ATP was 4 microM, and it increased to 15 microM in the presence of fructose 2,6-bisphosphate. The addition of 50 microM fructose 1,6-bisphosphate increased the KD for ATP to 5.9 microM. AMP increased the KD to 5.9 microM whereas 0.3 mM citrate decreased the KD for ATP to about 2 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
研究了2,6 - 二磷酸果糖对纯化的猪肾磷酸果糖激酶激活的影响，该影响是果糖6 - 磷酸、ATP和柠檬酸浓度的函数。在2 mM ATP存在下，纯化的酶几乎完全被抑制。添加20 nM 2,6 - 二磷酸果糖可逆转这种抑制作用，并恢复超过80%的活性。在没有2,6 - 二磷酸果糖的情况下，反应对果糖 - 6 - 磷酸呈S形依赖关系。在1.5 mM ATP存在下，添加10 nM 2,6 - 二磷酸果糖可使果糖6 - 磷酸的半最大反应浓度（K0.5）从3 mM降至0.4 mM。这些结果清楚地表明，2,6 - 二磷酸果糖增加了酶对果糖6 - 磷酸的亲和力，并降低了ATP的抑制作用。在2,6 - 磷酸果糖存在下，柠檬酸的抑制程度也显著降低。在凝胶过滤研究中，检测了磷酸果糖激酶的各种效应物对ATP和果糖6 - 磷酸与酶结合的影响。发现肾磷酸果糖激酶每摩尔酶结合5.6摩尔果糖6 - 磷酸，这相当于四聚体酶每个亚基约一个位点。果糖6 - 磷酸的解离常数（KD）为13 μM，在0.5 mM ATP存在下增加到27 μM。添加0.3 mM柠檬酸也使果糖6 - 磷酸的KD增加到约40 μM。10 μM的AMP将KD降至5 μM，添加2,6 - 二磷酸果糖将果糖6 - 磷酸的KD降至0.9 μM。添加这些化合物不影响与酶结合的果糖6 - 磷酸的最大量，这表明这些化合物的结合位点可能靠近但不与果糖6 - 磷酸结合位点相同。该酶每摩尔最多结合约12.5摩尔ATP，相当于每个亚基3摩尔。对ATP亲和力最高的位点的KD为4 μM，在2,6 - 二磷酸果糖存在下增加到15 μM。添加50 μM 1,6 - 二磷酸果糖使ATP的KD增加到5.9 μM。AMP将KD增加到5.9 μM，而0.3 mM柠檬酸将ATP的KD降低到约2 μM。（摘要截短至400字）
