Self-stabilization of the energy charge in a reconstituted enzyme system containing phosphofructokinase.

The self-stabilization of thc cnergy charge and of ATP was investigated in an open reconstituted enzyme system containing phosphofructokinase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase. The experiments were performed in a stirred flow-through reactor containing gel-entrapped enzymes. The dynamics of the system were analyzed theoretically by a model based on the kinetic properties of the individual enzymes. The energy charge was identified as one of the essential variables of the system. According to the theoretical prediction, homoeostasis of the energy charge was observed experimentally when either the maximal activity of phosphofructokinase, the energy charge of the influx solution or the flow rate through the reaction chamber was varied. It is shown that the efficiency of stabilization of the energy charge is related to the occurrence of alternative stationary states. The energy charge, as introduced by D. E. Atkinson [I, 21, is widely accepted as an important quantity for the coordination and regulation of energy-supplying and energyutilizing metabolic pathways. Cells in general are capable of maintaining a high internal energy charge at a wide range of external and internal loads [3,4]. Theoretical considerations based on the concept of nonequilibrium thermodynamics [5] led to the conclusion that the homoeostasis of the cellular ATP level is the result of nonlinear interactions arising from the underlying network of chemical reactions and from allosteric relationships. Experimental investigations of the mechanisms leading to energy stabilization in the cell are scarce. In particular, it is not completcly clear whether the energy charge as such or a related parametcr, e.g. the ATP concentration or the phosphorylation potential of the adenine nucleotides, are actually controlled. The mechanisms responsible for the intracellular stabilization of the energy charge or of the ATP level have not hitherto been satisfactorily clarified by adequate experimcntal approaches. In this paper the phenomenon of homoeostasis of the energy charge and of the ATP level is investigated in a reconstituted enzyme system containing phosphofructokinase, pyruvate kinase, adcnylate kinase, and glucose-6-phosphate isomerase operating under open conditions far from the thermodynamic equilibrium. The nonlinear structure gives rise to the simultaneous occurrence of several steady states expressed by alternative reactant patterns and substrate flow