Mechanistic basis for the allosteric activation of mitochondrial glutaminase C, a key driver of glutamine metabolism in cancer cells.

The dependence of cancer cells on glutamine metabolism, the most abundant amino acid in plasma, has been observed in many highly aggressive and deadly cancers including pancreatic cancer, triple-negative breast cancer, and glioblastoma. The mitochondrial enzyme glutaminase C (GAC) catalyzes the hydrolysis of glutamine to glutamate, the first step in glutamine metabolism, highlighting GAC as a potentially important therapeutic target. GAC acquires maximal catalytic activity upon binding to anionic activators like inorganic phosphate. To delineate the mechanism of GAC activation, we used the tryptophan substitution of tyrosine 466 in the catalytic site of the enzyme as a fluorescence reporter for glutamine binding in the presence and absence of phosphate. We show that in the absence of phosphate, glutamine binding to the GAC (Y466W) tetramer exhibits positive cooperativity. A high-resolution X-ray structure of tetrameric GAC (Y466W) bound to glutamine suggests that cooperativity in substrate binding is coupled to tyrosine 249, located at the edge of the catalytic site (i.e. designated the 'lid'), adopting two distinct conformations. In one dimer within the GAC tetramer, the lids are open and glutamine binds weakly, whereas, in the adjoining dimer, the lids are closed over the substrates resulting in higher affinity interactions. When crystallized in the presence of glutamine and phosphate, all four subunits of the GAC (Y466W) tetramer have bound glutamine with closed lids. Glutamine now binds with high affinity to each subunit, which then undergo simultaneous catalysis. These findings show how the regulated transitioning of GAC between different conformational states ensures maximal catalytic activity is reached in cancer cells only when an allosteric activator is available.