IMPLICATIONS OF MACROMOLECULAR CROWDING FOR SIGNAL-TRANSDUCTION AND METABOLITE CHANNELING

The effect of different total enzyme concentrations on the flux throug h the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase sy stem (PTS) in vitro aas determined by measuring PTS-mediated carbohydr ate phosphorylation at different dilutions of cell-free extract of Esc herichia coil, The dependence of the flux on the protein concentration was more than linear but less than quadratic. The combined flux-respo nse coefficient of the four enzymes constituting the glucose PTS decre ased slightly from values of approximate to 1.8 with increasing protei n concentrations in the assay. Addition of the macromolecular crowding agents polyethylene glycol (PEG) 6000 and PEG 35000 led to a sharper decrease in the combined flux-response coefficient, in one case to val ues of approximate to 1. PEG 6000 stimulated the PTS flux at lower pro tein concentrations and inhibited the flux at higher protein concentra tions, with the transition depending on the PEG 6000 concentration, Th is suggests that macromolecular crowding decreases the dissociation ra te constants of enzyme complexes, High concentrations of the microsolu te glycerol did not affect the combined flux-response coefficient. The data could be explained with a kinetic model of macromolecular crowdi ng in a two-enzyme group-transfer pathway. Our results suggest that, b ecause of the crowded environment in the cell, the different PTS enzym es form complexes that live long an the time-scale of their turnover. The implications for the metabolic behavior and control properties of the PTS, and for the effect of macromolecular crowding on nonequilibri um processes, are discussed.