The quaternary structure of pyruvate kinase type 1 from Escherichia coli at low nanomolar concentrations

Pyruvate kinase (PK) is the key control point of glycolysis—the biochemical pathway central to energy metabolism and the production of precursors used in biosynthesis. PK type 1 from Escherichia coli (Ec-PK1) is activated by both fructose-1,6-bisphosphate (FBP) and its substrate, phosphoenol pyruvate (PEP). To date, it has not been possible to determine whether the enzyme is tetrameric at the low concentrations (i.e. low nM range) used to study the steady-state kinetics, or assess whether its allosteric effectors alter the oligomeric state of the enzyme at these concentrations. Employing the new technique of analytical ultracentrifugation with fluorescence detection we have, for the first time, shown that the KD4–2 for Ec-PK1 is in the subnanomolar range, well below the concentrations used in kinetic studies. In addition, we show that, unlike some other PK isoenzymes, the modulation of oligomeric state by the allosteric effectors FBP and PEP does not occur at a concentration of 10 nM or above.