Bidirectional Allosteric Ligand Regulation in a Central Glycolytic Enzyme.

Allosteric regulation enables fine-tuned control of enzyme activity in response to cellular signals, yet its molecular basis often remains unclear. Phosphofructokinase-1 (PFK), the highly conserved, rate-limiting glycolytic enzyme, is a paradigm for Monod-Wyman-Changeux allosteric kinetics. However, X-ray crystal structures of bacterial PFK orthologs in distinct ligand-bound states do not show the consistent, concerted structural rearrangements expected for classical "relaxed" and "tense" states, revealing a decades-long disconnect between structure and function. We resolve this paradox by integrating biophysical and computational approaches to show that activator and inhibitor binding to the same allosteric pocket differentially reweight the conformational ensemble of Escherichia coli PFK. Activator binding enriches conformational substates that preorganize the catalytic site, whereas inhibitor binding upweights apo-like, catalytically incompetent substates. These findings establish an ensemble-based mechanism for PFK regulation and provide an energetic framework for understanding the expanded allosteric architecture of eukaryotic PFK orthologs.