Phosphofructokinase from rabbit skeletal muscle is an allosteric enzyme as a key regulatory site in the glycolysis sequence. The regulatory characteristics of phsophofructokinase were studied by using coupled assay methods.
Phosphofructokinase activity was inhibited by high concentrations of substrate ATP, and the degree of ATP inhibition was dependent on the concentrations of the second substrate fructose 6-phosphate, i.e. the lower level of fructose 6-phosphate was, the higher degree of ATP inhibition appeared. Initial rate profile as a function of fructose 6-phosphate was sigmoidal, but at the very low concentrations of ATP, hyperbolic nature was also observed.
Under the high concentration of ATP and/or low concentration of fructose 6-phosphate, the enzyme activity was affected by the presence of creatine phosphate, i.e. inhibition of enzyme activity and stimulation of ATP inhibition were observed by the increasing concentrations of creatine phosphate. The extent of substrate inhibition by ATP and also the sigmoidicity as a function of fructose 6-phosphate were increased remarkably by the presence of creatine phosphate.
ITP could act as substrate but could not cause substrate inhibition, in contrast with ATP. When ITP was used as substrate, creatine phosphate did not have any effect on the enzyme activity.
It is believed that creatine phosphate act as a modulator in the phosphofructokinase system, and it seems that the regulatory role of creatine phosphate is very closely related to the ATP inhibition.
Based on these results from kinetic experiments and some chemical modifications, the regulatory role of creatine phosphate in phosphofructokinase reaction was suggested in relation to their possible role in glycolytic regulation.
Phosphofructokinase 는 해당작용 계에 속하는 효소로, PH, 온도, 저해제, 촉진제등 여러요소에 의해 조절을 받는다. Creatine phosphate 가 토끼근육에 존재하는 Phosphofructokinase 에 작용하는 조절효과를 연구하였다.
조절작용이 현저하게 나타나는 PH 7.0 에서는 기질의 하나인 ATP가 고농도로 존재할때 효소가 저해를 받으며, 이 저해정도는 또 하나의 기질인 fructose 6 -phosphate 의 농도가 낮을수록 커진다. fructose 6 -phosphate 의 농도에 대한 초기속도곡선은 ATP 의 농도가 낮을 때는 쌍곡선을 그리나 ATP의 농도가 높아질수록 S 자 곡선의 형태로 나타났다.
Phosphofructokinase 가 ATP 에 의한 저해작용을 받는 조건하에서 실험한 결과, creatine phosphate 는 phosphofructokinase 의 활성을 저해할 뿐 아니라 ATP 에 의한 저해정도를 증가시키고 효소의 ATP 저해작용에 대한 감수성을 크게하며, fructose 6 -phosphate 농도에 대한 S 자 곡선의 변화도를 증가시켰다. 따라서 creatine phosphate 의 기능은 ATP에 의한 저해작용과 깊은 연관이 있음을 알 수 있다.
Phosphofructokinase 의 기질 특이성을 조사한 실험과 효소에 대한 화학변화제 처리 실험결과에 의하면, creatine phosphate 는 기질들의 부착장소에는 작용하지 않을 것으로 생각되며, PH- 활성도의 관계에도 전혀 영향을 주지 않았다.
기질로서는 작용하나 저해제로서는 작용하지 못하는 ITP를 기질로 한 실험에서는 ATP 의 경우와는 달리 creatine phosphate 가 전혀 저해작용을 하지 않았다.
이상의 실험결과들로 보아, creatine phosphate 는 저해제로서의 ATP 부착위치와는 다른 곳에 부착하여 ATP 와 기질저 해부착 장소와의 친화성을 증가시켜 부착을 촉진함으로써 앞서 설명한 phosphofructokinase 에 대한 조절효과를 나타내는 것으로 생각된다.
