Regulation of Enzymes in situ

Abstract

For an accurate understanding of metabolic regulation at the enzyme level it is important to ascertain to what extent mechanisms of modulation of enzyme activity observed in vitro can have a physiologically significant counterpart in vivo. As an approximation to this ultimate goal we have recently succeeded in revealing the activity of phosphofructokinase in E. coli by a toluene-freezing permeabilization treatment that makes the enzyme directly accessible to external substrates and other small molecules while leaving it within a damaged cell membrane (or at least within the cell wall), conditions which we call in situ. The permeabilization method has also been adapted to yeast.

E. Coli phosphofructokinase in situ exhibits moderate cooperativity with respect to fructose-6-P (n_H up to 2.0), rather low affinity for ATP (with a Km up to 1 mM when saturated with F6P), activation by ADP, and allosteric inhibition, within the physiological range of concentrations, by phosphoenolpyruvate and high ATP. This behavior of the enzyme in situ at concentrations of the effector metabolites as those reported in intact cells in glycolytic and gluconeogenic conditions could account for the changes of phosphofructokinase activity needed for metabolic regulation in vivo.

The allosteric behavior of yeast pyruvate kinase in situ is identical to that in vitso. Preliminary observations with yeast fructose-1,6-diphosphatase in situ suggest that the Km in vivo may be markedly higher than the very low values exhibited by the enzyme in vitso.

It is suggested that this approach to the study of enzymes in situ could be particularly important for interconvertible enzyme systems, because of the protein-protein interactions essentially involved.