Dynamic properties of a phosphofructokinase/pyruvate kinase system. Experiments in vitro using the substrate-stat technique.

It is known from theoretical work that very simple enzyme systems may exhibit non-linear dynamic properties like those found in vivo. To realize such a system experimentally, an ATP-producing reaction (pyruvate kinase) was coupled with an ATP-consuming reaction (phosphofructokinase). The substrate-stat technique has been adapted to characterize each single enzyme as well as to follow up one enzyme in the coupled system. If the plots of both pyruvate kinase activity versus [ADP] and phosphofructokinase activity versus [ATP] are hyperbolic, the coupled system approaches a unique steady state as predicted from the single characteristics. Under assay conditions where phosphofructokinase is inhibited by ATP, its characteristics as found in a single assay and in the coupled system are different. For a system with ATP-inhibited phosphofructokinase, a paradoxical behaviour is predicted and is demonstrated experimentally. If the total pyruvate kinase activity is increased over a certain limit, the system is switched from a low [ATP], high-activity steady state into a high [ATP] low-activity steady state.