Alanine Rewires the Communication Pathways Established During the Allosteric Activation of Liver Pyruvate Kinase by...

Allostery enables enzymes to transmit information from distant ligand-binding sites to catalytic centers through residue-to-residue communication pathways. Human liver pyruvate kinase (hL-PYK) is a prototypical system that integrates activating input from fructose-1,6-bisphosphate (FBP) and inhibitory input from alanine, yet how these opposing signals propagate through the protein remains unclear. Here, we combine multimicrosecond molecular dynamics of the hL-PYK tetramer with residue-residue contact analysis and network/path mapping to define the underlying communication pathways. Contact-change correlations show that alanine antagonizes FBP by reversing many FBP-induced rearrangements. Difference contact network analysis and path enumeration reveal that FBP and alanine rewire information flow along partially overlapping but distinct routes between the allosteric and active sites; residues with high path degeneracy, acting as hubs, emerge as key conduits and include positions that overlap with prior mutational hotspots while also suggesting new sites for experimental testing. Residue community analysis further demonstrates intercommunity edges that switch sign or weaken upon alanine binding, indicating inhibition via residue-residue contact network rerouting. Together, these results support a mechanistic model in which alanine rewires the communication pathways established during FBP-mediated activation of hL-PYK and provide concrete hypotheses for modulating enzyme activity with allosteric therapeutics.