Equilibrium relations between pyridine nucleotides and adenine nucleotides and their roles in the regulation of metabolic processes

Abstract

The differences between the redox states of the NAD-and NADP-couples in the cytoplasm have been measured. The ratio [free NADP]/[free NADPH 2] was found to be about 100,000 times smaller than the ratio [free NAD]/[NADH 2]. These differences can be explained on the basis of simple physicochemical principles. They are essentially the result of thermodynamic equilibria. The position of these depends on the equilibrium constants of a number of highly active, readily reversible enzymes and the concentration of the substrates and products of these enzymes. The crucial feature is the fact that the NAD- and NADP-couples share substrates. This sharing represents links between the two couples in the cytoplasm and through these links the order of magnitude of the differences in the redox state is fixed. Analogous explanations can be visualized for the origin of the large differences between the redox states of the cytoplasm and mitochondrial NAD-couples but these involve assumptions regarding the distribution of metabolites between cytoplasm and mitochondria which can as yet not be reliably tested by experiment. There is a need for developing methods for measuring the distribution in situ. The redox states of the cytoplasmic pyridine nucleotide couples are also linked to the phosphorylation state of the adenine nucleotide, i.e., to the ratio [ATP]/[ADP] [Pi] of the cytoplasm, again by enzyme systems—the glyceraldehyde phosphate dehydrogenase plus 3-phosphoglycerate kinase—which establish equilibria. There is thus a network of equilibria, or more correctly near-equilibria, which is ultimately regulated by the degree of phosphorylation of the adenine nucleotide system, i.e., by the respiratory chain: the cytoplasmic adenine nucleotide system is directly linked to the redox state of the cytoplasmic NAD-couple which in turn is linked to the redox state of the cytoplasmic NADP-couple and that of the two mitochondral couples. The network of near-equilibria in which the pyridine and adenine nucleotides participate is likely to be a fundamental component of the energy-transforming mechanisms in the liver cell. It establishes a basic level of the redox states of the two pyridine nucleotide couples in the two main cell compartments where the energy-transforming mechanisms are located and it links the redox states to the supply of ATP. It sets the cytoplasmic level of the NAD-couple to be suitable for both glycolysis and gluconeogenesis. It sets the cytoplasmic redox state of the NADP-couple at a much more reduced level so as to be effective in reductive syntheses such as that of fatty acids, the redox potential being about 150 mV more negative than that of the NAD-couple. This network is one of thermodynamic equilibria whereas living cells, of course, do not represent equilibria but steady states. But the fact that thermodynamic equilibrium of a cell is synonymous with death does not imply that equilibria cannot play an important part in the organization of the chemical cell dynamics. Equilibria form a basic framework upon which virtually irreversible processes are superimposed. These flow through the equilibria, more or less upsetting them and modifying the strict equilibria to steady states. It is the flux through the network of equilibria which causes the degradation of food and the synthesis of the cell material—both processes which, as a whole, are irreversible. The framework of equilibria may be looked upon as a component of regulatory mechanisms: as irreversible processes flow through and mildly upset the equilibria, they automatically set in motion steps which reestablish the ‘resting’ state.