Can Macromolecular Crowding Help Regulate Glutamate Dehydrogenase Activity?

Glutamate dehydrogenase (GDH) is an important mitochondrial enzyme that is positioned at the intersection of several central metabolic pathways. Since this enzyme influences the flux of crucial metabolites, GDH activity is tightly controlled by a complex network of allosteric effectors, and disruption of this regulation has been correlated with a growing list of diseases. To better understand how the crowded environment and pH fluctuations of the mitochondrial matrix contribute to the fine-tuning of GDH regulation, Michaelis-Menten kinetics were measured in the presence of both synthetic and protein crowding agents. The results show a pH-dependent decrease in the GDH activity regardless of crowder identity. Specifically, macromolecular crowding favors the closed GDH conformation, thereby slowing product release. In addition, the presence of dextran increases the pK _a of a crucial lysine residue on GDH, while glucose, its small-molecule counterpart, does not. These kinetic results, together with Eyring plots and classical molecular dynamics simulations, suggest that excluded volume effects promote an abortive GDH-glutamate-NADH complex at lower pH (∼7). Under these conditions, crowding abrogates leucine activation but does not diminish GTP inhibition. Together, these findings indicate a complex interrelationship among macromolecular crowding, pH, and allosteric effectors to finely tune the GDH activity.