There is a growing interest in rationally designing allosteric drugs. However, before initiating the costly steps of drug development, new tools are needed to confirm that targeted allosteric regulations of individual enzymes result in desired physiological outcomes. For example, allosteric activation of human liver pyruvate kinase (hLPYK) might be useful for activating glycolysis in the diabetic liver, thus reducing hepatic glucose production. In detailed in vitro studies that used a linked-function definition to describe allosteric mechanisms, we identified individual point mutations that: 1) prevent hL-PYK from binding the allosteric inhibitor, alanine; 2) prevent the inhibitory covalent modification, protein phosphorylation; or 3) mimic activation by the allosteric activator, fructose-1,6-bisphosphate. The combination of all three point mutations results in an enzyme that constitutively binds the substrate, PEP, with high affinity, similar to the fully activated wild type enzyme. Furthermore, this modified enzyme does not respond to regulation by allosteric effectors or phosphorylation. We envision that in vivo gene editing to place well-characterized point mutations in model systems will be useful for confirming the hypothesized roles of allosteric regulations in controlling whole animal processes. As we move our own research towards those gene edited designs, our current progress demonstrates that mutations that remove or mimic allosteric regulators are useful tools to customize enzyme substrate affinities.

Mutational mimics of allosteric effectors to customize enzyme-substrate affinity

254th National Meeting (2017)

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