Boscá L, Aragón J J, Sols A
J Biol Chem. 1985 Feb 25;260(4):2100-7.
Two approaches have been used to study the allosteric modulation of phosphofructokinase at physiological concentration of enzyme; a "slow motion" approach based on the use of a very low Mg2+/ATP ratio to conveniently lower Vmax, and the addition of polyethylene glycol as a "crowding" agent to favor aggregation of diluted enzyme. At 0.6 mg/ml muscle phosphofructokinase exhibited a drastic decrease in the ATP inhibition and the concomitant increase in the apparent affinity for fructose-6-P, as compared to a 100-fold diluted enzyme. Similar results were obtained with diluted enzyme in the presence of 10% polyethylene glycol (Mr = 6000). Results with these two approaches in vitro were essentially similar to those previously observed in situ (Aragón, J. J., Felíu, F. E., Frenkel, R., and Sols, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6324-6328), indicating that the enzyme is strongly dependent on homologous interactions at physiological concentrations. With polyethylene glycol it was observed that within the physiological range of concentration of substrates and the other positive effectors, fructose-2,6-P2 still activates the liver phosphofructokinase although it no longer significantly affects the muscle isozyme. In the presence of polyethylene glycol, muscle phosphofructokinase can approach its maximal rate even in the presence of physiologically high concentrations of ATP. Three minor activities of muscle phosphofructokinase have been studied at high enzyme concentration: the hydrolysis of MgATP (ATPase) and fructose-1,6-P2 (FBPase), produced in the absence of the other substrate, and the reverse reaction from MgADP and fructose-1,6-P2. The kinetic study of these activities has allowed a new insight into the mechanisms involved in the modulation of phosphofructokinase activity. The binding of (Mg)ATP at its regulatory site reduces the ability of the enzyme to cleave the bond of the terminal phosphate of MgATP at the substrate site. The positive effectors (Pi, cAMP, NH+4, fructose-1,6-P2, and fructose-2,6-P2) decrease the inhibitory effect of MgATP. Citrate and fructose-2,6-P2 both act as mechanistically "secondary" effectors in the sense that citrate does not inhibit and fructose-2,6-P2 does not activate the FBPase activity, requiring both the presence of ATP to affect the enzyme activity. In conclusion it appears that the regulatory behavior of mammalian phosphofructokinases is utterly dependent on the fact of their high concentrations in vivo.
人们采用了两种方法来研究生理浓度下磷酸果糖激酶的变构调节；一种是“慢动作”方法，即利用极低的Mg2+/ATP比值来方便地降低Vmax，另一种是添加聚乙二醇作为“拥挤”剂，以促进稀释酶的聚集。与稀释100倍的酶相比，在0.6mg/ml时，肌肉磷酸果糖激酶的ATP抑制作用急剧降低，同时对果糖-6-磷酸的表观亲和力增加。在10%聚乙二醇（Mr = 6000）存在下的稀释酶也得到了类似的结果。这两种体外方法的结果与之前原位观察到的结果基本相似（阿拉贡，J. J.，费利乌，F. E.，弗伦克尔，R.，和索尔斯，A.（1980年）美国国家科学院院刊77，6324 - 6328），表明该酶在生理浓度下强烈依赖同源相互作用。使用聚乙二醇观察到，在底物和其他正效应物的生理浓度范围内，果糖-2,6-二磷酸仍能激活肝脏磷酸果糖激酶，尽管它对肌肉同工酶的影响不再显著。在聚乙二醇存在下，即使在生理浓度较高的ATP存在下，肌肉磷酸果糖激酶也能接近其最大反应速率。在高酶浓度下研究了肌肉磷酸果糖激酶的三种次要活性：在没有其他底物时产生的MgATP（ATP酶）和果糖-1,6-二磷酸（FBP酶）的水解，以及由MgADP和果糖-1,6-二磷酸的逆向反应。对这些活性的动力学研究为深入了解磷酸果糖激酶活性调节机制提供了新的视角。（Mg）ATP在其调节位点的结合降低了酶在底物位点切割MgATP末端磷酸键的能力。正效应物（Pi、cAMP、NH4+、果糖-1,6-二磷酸和果糖-2,6-二磷酸）降低了MgATP的抑制作用。柠檬酸和果糖-2,6-二磷酸在机制上都作为 “二级” 效应物，因为柠檬酸不抑制且果糖-2,6-二磷酸不激活FBP酶活性，两者都需要ATP的存在才能影响酶活性。总之，哺乳动物磷酸果糖激酶的调节行为似乎完全依赖于它们在体内的高浓度这一事实。
