Skeletal muscle phosphofructokinase activity examined under physiological conditions in vitro

The in vitro activity of skeletal muscle phosphofructokinase (PFK) was determined over the full physiological range of citrate concentrations. Enzyme aggregation was enhanced with a crowding agent, as the regulatory properties of PFK are altered with dilution. Cuvette conditions simulated concentrations of effectors and substrates during rest, moderate aerobic exercise, and intense aerobic exercise in human skeletal muscle. Citrate inhibition was not eliminated with enhanced enzyme aggregation, but activity was improved at all citrate concentrations. Maximal PFK activity with no citrate present was 0.27 +/- 0.01 mumol.min-1.mg-1 protein with resting effectors and 1.64 +/- 0.07 and 7.15 +/- 0.52 mumol.min-1.mg-1 protein with moderate aerobic and intense aerobic effector levels, respectively. Under resting conditions, PFK activity decreased to 49% of maximal when citrate was increased from 0 to 0.15 mM and only a small further inhibition to 43% occurred at 0.5 mM. Citrate was a less potent inhibitor under both exercise conditions with the sharpest decline to 72-77% of maximal activity at 0.15 mM followed by a slower decline to 65-70 and 53% activity at 0.25 and 0.5 mM citrate, respectively. The present in vitro measurements predict that alterations in citrate around concentrations normally reported in resting and exercising muscle would have little effect on flux through PFK. Therefore, the generally accepted concept that citrate is a potent inhibitor of PFK in all physiological situations has been exaggerated.