Directional regulation of cytosolic PEPCK catalysis is mediated by competitive binding of anions.

Phosphoenolpyruvate carboxykinase (PEPCK) is a well-characterized enzyme involved in primary glucose metabolism, responsible for catalyzing one of the key steps of gluconeogenesis. It is well demonstrated that PEPCK can efficiently catalyze the reversible interconversion of oxaloacetic acid (OAA) to phosphoenolpyruvate (PEP) in vitro, but the enzyme is typically ascribed a metabolic role that requires preferential catalysis in the direction of PEP synthesis in vivo. Here we present structural and functional data that demonstrate the preferential synthesis of PEP from OAA catalyzed by PEPCK in vivo is facilitated by anion-mediated enzyme inhibition that reduces enzyme activity more significantly in the direction of OAA synthesis than in the direction of PEP synthesis. From our studies we conclude that the specific binding of small, ubiquitous anions like chloride, present in millimolar concentrations under normal cellular conditions allows for metabolic control by restricting PEPCK to function in the direction of PEP synthesis.

A structural perspective on the temperature-dependent activity of enzymes.

Enzymes are biomolecular catalysts whose activity varies with temperature. Unlike for small-molecule catalysts, the structural ensembles of enzymes can vary substantially with temperature, and it is in general unclear how this modulates the temperature dependence of activity. Here multi-temperature X-ray crystallography was used to record structural changes from -20°C to 40°C for a mesophilic enzyme in complex with inhibitors mimicking substrate-, intermediate-, and product-bound states, representative of major complexes underlying the kinetic constant k c a t . Both inhibitors, substrates and catalytically relevant loop motifs increasingly populate catalytically competent conformations as temperature increases. These changes occur even in temperature ranges where kinetic measurements show roughly linear Arrhenius/Eyring behavior where parameters characterizing the system are assumed to be temperature independent. Simple analysis shows that linear Arrhenius/Eyring behavior can still be observed when the underlying activation energy / enthalpy values vary with temperature, e.g., due to structural changes, and that the underlying thermodynamic parameters can be far from values derived from Arrhenius/Eyring model fits. Our results indicate a critical role for temperature-dependent atomic-resolution structural data in interpreting temperature-dependent kinetic data from enzymatic systems.

The H163A mutation unravels an oxidized conformation of the SARS-CoV-2 main protease.

The main protease of SARS-CoV-2 (Mpro) is an important target for developing COVID-19 therapeutics. Recent work has highlighted Mpro's susceptibility to undergo redox-associated conformational changes in response to cellular and immune-system-induced oxidation. Despite structural evidence indicating large-scale rearrangements upon oxidation, the mechanisms of conformational change and its functional consequences are poorly understood. Here, we present the crystal structure of an Mpro point mutant (H163A) that shows an oxidized conformation with the catalytic cysteine in a disulfide bond. We hypothesize that Mpro adopts this conformation under oxidative stress to protect against over-oxidation. Our metadynamics simulations illustrate a potential mechanism by which H163 modulates this transition and suggest that this equilibrium exists in the wild type enzyme. We show that other point mutations also significantly shift the equilibrium towards this state by altering conformational free energies. Unique avenues of SARS-CoV-2 research can be explored by understanding how H163 modulates this equilibrium.