BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF MURINE AND HUMAN GLUTAMINASE C

The classic biochemical model of metabolic enzymes as globular and particulate catalytic machines has been challenged in the last decades, with the development of top-of-the-line structural biology tools like single-particle Cryo-EM and Cryo-ET. New arrangements of enzymes intracellularly in rings, rods, foci, and filaments have demonstrated the existence of a super-quaternary level of protein organization. These assemblies have properties that their protomers do not, a kind of emergent property, such as the creation of new active sites, enzymatic inactivation or superactivation, substrate-specificity switches, regulation of cell and organelle shape, and others. Glutaminase C (GAC) reversibly forms filaments (fGAC) during glutamine deprivation or inorganic phosphate (Pi) addition. These filaments elongate mitochondria and impair mitophagy. Additionally, glutaminase drives glutaminolysis, converting glutamine to glutamate for energy and biosynthesis. This metabolic pathway is frequently upregulated in cancer, fueling tumor cell proliferation. To elucidate the structural basis of this filamentation and its broader metabolic role, truncated murine (mGAC) and human GAC (hGAC) isoform constructs were expressed in E. coli and purified to homogeneity via affinity, ion-exchange, and size-exclusion chromatography. Enzymatic assays confirmed Pi-dependent allosteric activation, evidenced by a reduction in the Km values for glutamine (up to 7 mM) and a nearly tenfold increase in catalytic efficiency upon the addition of 20 mM Pi. Dynamic light scattering demonstrated stable fGAC formation in the presence of Pi, resulting in an increased hydrodynamic radius (>20 nm) after two hours of incubation. Glutamine and glutamate modulated filamentation dynamics, producing intermediate species with different hydrodynamic radii, which suggests substrate- and product-dependent structural plasticity. Initial cryo-EM analysis of mGAC and hGAC revealed a canonical tetramer-based filament architecture and conformational heterogeneity consistent with multiple oligomeric intermediates based on 2D classification. Filamentation could serve additional functions, such as acting as an interaction hub for other proteins. In this regard, an interaction network assembled via BioGRID and structurally modeled with AlphaFold 3 revealed that hGAC predominantly binds to enzymes within interconnected metabolic pathways.

This work was supported by São Paulo Research Foundation (FAPESP) grants: 2024/22806-1 and 2021/05726-6.