Protein-metabolite interactions are critical for the rapid, protein-level regulation of metabolic enzymes in response to cellular changes. While extensively studied in central carbon metabolism, their prevalence in nitrogen metabolism is less understood. A prime example is glutamine synthetase (GS), a key enzyme in nitrogen metabolism vital for cell growth and detoxification. GS dysregulation is implicated in significant diseases, from cancer to neurodegenerative disorders like hepatic encephalopathy and Alzheimer's. Despite its importance, protein-level regulation of GS is poorly characterized, largely due to a dearth of identified allosteric regulators capable of selectively modulating its activity. To identify novel regulators, we screened a library of over 800 endogenous metabolites for their impact on GS Michaelis-Menten constants using an NADH-coupled continuous assay, with a counter-screen to ensure specificity. This effort led to the discovery of neopterin as an inhibitor of GS activity. We are currently investigating the precise nature of this interaction through steady-state kinetics and cryo-electron microscopy (cryoEM). Elucidating the neopterin binding site on GS will determine if its inhibition is allosteric, a finding that could significantly broaden the therapeutic strategies for targeting GS beyond existing orthosteric inhibitors.
Jacobs et al. (2026) studied this question.