Many studies of allosteric mechanisms use limited numbers of mutations to test whether residues play “key” roles. However, if a large percentage of the protein contributes to allosteric function, mutating any residue would have a high probability of modifying allostery. Thus, a predicted mechanism that is dependent on only a few residues could erroneously appear to be supported. We used whole‐protein alanine‐scanning mutagenesis to determine which amino acid sidechains of human liver pyruvate kinase (hL‐PYK; approved symbol PKLR) contribute to regulation by fructose‐1,6‐bisphosphate (Fru‐1,6‐BP; activator) and alanine (inhibitor). Each nonalanine/nonglycine residue of hL‐PYK was mutated to alanine to generate 431 mutant proteins. Allosteric functions in active proteins were quantified by following substrate affinity over a concentration range of effectors. Results show that different residues contribute to the two allosteric functions. Only a small fraction of mutated residues perturbed inhibition by alanine. In contrast, a large percentage of mutated residues influenced activation by Fru‐1,6‐BP; inhibition by alanine is not simply the reverse of activation by Fru‐1,6‐BP. Moreover, the results show that Fru‐1,6‐BP activation would be extremely difficult to elucidate using a limited number of mutations. Additionally, this large mutational data set will be useful to train and test computational algorithms aiming to predict allosteric mechanisms.

"Allosteric" was first introduced to mean the other site (i.e., a site distinct from the active or orthosteric site), an adjective for "regulation" to imply a regulatory outcome resulting from ligand binding at another site. That original idea outlines a system with two ligand-binding events at two distinct locations on a macromolecule (originally a protein system), which defines a four-state energy cycle. An allosteric energy cycle provides a quantifiable allosteric coupling constant and focuses our attention on the unique properties of the four equilibrated protein complexes that constitute the energy cycle. Because many observed phenomena have been referenced as "allosteric regulation" in the literature, the goal of this work is to use literature examples to explore which systems are and are not consistent with the two-ligand thermodynamic energy cycle-based definition of allosteric regulation. We emphasize the need for consistent language so comparisons can be made among the ever-increasing number of allosteric systems. Building on the mutually exclusive natures of an energy cycle definition of allosteric regulation versus classic two-state models, we conclude our discussion by outlining how the often-proposed Rube-Goldberg-like mechanisms are likely inconsistent with an energy cycle definition of allosteric regulation.