Our experimental design will draw a contrast to two state mechanism. Two state mechanisms of allostery define two end state conformations (or two end state ensembles), leaving the steps for the interconversion of these two end states as the focus of many modern studies. However, a focus on the kinetics of conformational change (i.e., “conetics” to distinguish from other kinetic processes), de-emphasizes the equilibrium nature of allostery.
In contrast to a focus on conetics, for a K-type system, allostery is a comparison of the affinity of a macromolecule (e.g., an enzyme) for a ligand (e.g., a substrate) in the absence versus presence of an allosteric effector, recognizing in our example that the effector binds to a site distinct from the active site. Binding of substrate to the enzyme is an equilibrium event. Therefore, the comparison of two binding events (i.e., in the absence vs. presence of effector) is also an equilibrium process. This logic defines four enzyme states: 1) the free enzyme, 2) the enzyme/substrate complex, 3) the enzyme/effector complex, and 4) the ternary substrate/enzyme/effector complex. It follows that the dynamics that contribute to equilibrium allostery are the differences in the “wiggles and giggles” of the equilibrated protein states, identified by comparing the four enzyme states. A ratio of the enzyme-to-substrate binding constants in the absence vs. presence of effector defines the allosteric coupling constant (Qax) that can be converted into free energy.
Qax = Kia/Kia/x ,
where Kia is the binding constant for substrate (A) in the absence of effector, Kia/x is the binding constant for substrate when effector (X) concentration is sufficiently high to saturate the effector binding site. Importantly, because Qax is a ratio, the magnitude of the allosteric coupling is independent of how tightly the allosteric effector binds. Based on this, we use ligand analogues and large numbers of point mutations to distinguish in human liver pyruvate kinase (hLPYK) which protein/ligand interactions contribute to allostery from those that contribute to ligand binding.
After characterizing the allosteric regulation of mutations and analogues to evaluate the allosteric mechanism, our studies branch in many directions. Data have been used in computational analysis to test and validate those analysis. Mutations based on the mutational studies have been used in structural studies to gain deeper insights into those allosteric mechanisms. Finally, mutations that prevent or mimic the allosteric responses have been introduce into mouse genomes to evaluate the influence of the allosteric responses on metabolic regulation in mouse models.