Filament formation by glutaminase enzymes drives catalysis.

The mitochondrial glutaminase enzymes initiate glutaminolysis by catalyzing the hydrolysis of glutamine to glutamate, satisfying the metabolic requirements of aggressive cancers and thus representing potential therapeutic targets. However, the mechanisms underlying their allosteric regulation are poorly understood. It has been suggested that glutaminases form oligomeric filament-like structures essential for their activation. Here, we provide structural evidence for the ability of the glutaminase enzymes to form filaments upon substrate binding, and present the first cryo-EM structures of the human full-length glutaminase isozyme GLS2 that offer an unprecedented view of the mechanism responsible for catalyzing glutamine hydrolysis. The GLS2 structures reveal that the 'activation loop', a motif previously identified to regulate enzymatic activity, assumes a unique conformation and works together with a 'lid' that closes over the active site to 'lock in' the substrate glutamine. Tyrosine 251 of the GLS2 activation loop forms a cation-π interaction with Lysine 222 in the active site, which in turn enables a key catalytic residue, Serine 219, to undergo deprotonation for nucleophilic attack on the substrate. These findings further suggest that allosteric glutaminase inhibitors disrupt this interaction, which is critical for catalysis, while activators stabilize it. The GLS2 structures also show how the ankyrin repeats regulate different glutaminase isozymes.