(A) Native mass spectra of 10 µM PKM2 in 200 mM ammonium acetate at pH 6.8, in the absence of allosteric ligands (PKM2apo*), or in the presence of: 10 µM FBP (PKM2FBP), 300 µM Phe (PKM2Phe), 300 µM Phe followed by addition of 10 µM FBP (PKM2Phe + FBP) or 10 µM FBP followed by addition of 300 µM Phe (PKM2FBP + Phe). (B) Relative abundance of PKM2 monomers, dimers and tetramers obtained from the spectra shown in (A) by computing the area of the peaks corresponding to each of the three oligomeric states. Relative peak areas were calculated as a percentage of the total area given by all charge-state species in a single mass spectrum. (C) Deconvolved mass spectra of PKM2 tetrameric species in the absence of any added ligands (PKM2apo*) or presence of FBP (PKM2FBP). PKM2apo* has five distinct mass peaks, separated by approximately 340 Da (equivalent to the weight of FBP), corresponding to tetrameric PKM2apo*, and tetrameric PKM2 bound to 1, 2, 3 and 4 molecules of FBP, respectively. See Table 4 for the theoretical, and Table 5 for the experimentally measured masses of PKM2, FBP and their complexes. The spectrum of PKM2FBP contains a single peak, corresponding to tetrameric PKM2 bound to four molecules of FBP. (D) DTCCSHe distribution of PKM2apo*, PKM2FBP and PKM2FBP+Phe tetramers calculated from analyses of arrival time distribution measurements of PKM2 tetramer peaks (see Materials and methods). The plots at the bottom show the mean difference of the DTCCSHe distributions between the indicated liganded states. Grey shaded regions show the standard deviations of the distribution differences. (E) Change, over time, in the oligomeric state of PKM2 upon addition of sub-stoichiometric FBP and saturating Phe concentrations. Oligomerisation is reported as the ratio of the tetramer 32 + charge state peak relative to the dimer 22 + charge state peak, obtained from mass spectra of 10 µM PKM2 following addition of either 5 µM FBP, or 5 µM FBP and 400 µM Phe over the course of 20 min. The kinetics of tetramerisation were estimated from a two-state sigmoidal model (orange and blue dashed lines, see Materials and methods). In the legend, 0.5 FBP and 40 Phe indicate molar ratio of these ligands compared to PKM2.