Glutamate binding perturbs structure-function integrity of carbonic anhydrase and promotes its aggregation: Implications of dysregulated metabolite levels in neurodegeneration.

Metabolite dysregulation is increasingly recognized as a crucial factor in protein misfolding and aggregation underlying various neurodegenerative diseases. Among these, glutamate, the most abundant excitatory neurotransmitter, is strongly regulated under physiological conditions but reaches pathological concentrations in most of the neurodegenerative disorders. In this context, the present study investigated how glutamate modulates the structure-function integrity of carbonic anhydrase (CA), a neuronal enzyme implicated in brain homeostasis, pH regulation, and cognitive processes. Employing enzyme kinetics, spectroscopic and calorimetric assays, and molecular dynamics simulations, it was observed that glutamate significantly inhibits CA activity and disrupts its tertiary and secondary structure with reduced thermodynamic stability. The observed glutamate-induced alterations were associated with increased solvent exposure, structural fluctuations, and loss of compactness, resulting in pronounced aggregation of CA confirmed by light scattering measurements and electron microscopy. Moreover, calorimetric measurements revealed binding interactions between glutamate and CA, supporting a metabolite-driven mechanism of destabilization and aggregation. Altogether, the present findings demonstrate that elevated glutamate concentrations can alter structure-function integrity of CA. Although the physiological implications require further investigations, the study highlights the potential influence of metabolites on the conformational and functional integrity of enzymes under stress conditions.