Exploring the Energy Barriers Involved in Allosteric Transitions in Glutamate Dehydrogenase

Glutamate Dehydrogenase exhibits negative cooperativity, allosteric activation [by ADP], inhibition [by GTP] and substrate inhibition by glutamate. Each is thought to involve subunit interactions. Ligand induced changes in conformation occur with an energy barrier that must be overcome by energy from ligand binding. In negative cooperativity induced by NAD(P)+ binding, activity goes up but affinity for ligand goes down. We hypothesized that if the increased activity results from enhanced flexibility, raising the thermal energy of the system should overcome the energy barrier resulting in a single form of the enzyme with high activity and low affinity rather than the negative cooperativity. The temperature dependence of the non‐linear Lineweaver Burke plots resulting from negative cooperativity shows that the cooperativity disappears at temperatures above 40°C giving a form with high catalytic efficiency and low affinity, consistent with our hypothesis. At low temperature [15°C or below] the cooperativity also disappears suggesting the energy barrier to trigger the negative cooperativity is too high for ligand derived energy input to overcome. This approach has been extended to examination of both substrate inhibition and heterotropic allosteric regulation in this system to give information about associated energy barriers. Supported by NSF Grant MCB 0448905 to Ellis Bell