Interrogation of the human glutamate dehydrogenase antenna to elucidate its role in allosteric regulation and disease

Glutamate dehydrogenase (GDH) catalyzes the reversible oxidative deamination of L-glutamate using NAD(P)+ as a coenzyme. GDH is found in all living organisms, but only GDH from animals is highly allosterically regulated by a wide array of metabolites. The importance of this regulation is made evident by hyperinsulinism/hyperammonemia syndrome (HI/HA), where mutations cause GDH hyperactivity. Since only allosterically regulated forms of the enzyme contain an approximately 50-residue antenna domain, it suggests that the antenna is critical for allosteric regulation of GDH. To explore the role of the antenna in allostery, a series of antenna mutations were made, including removing the antenna and characterizing clinically relevant HI/HA mutations that have been previously identified in patients. Characterization of these mutants elucidates the role of the antenna and the mechanism of GDH hyperactivity in HI/HA. Prior to this investigation, a previous antenna-less GDH construct was generated that replaced the antenna with the short loop found in bacterial GDH. The chimeric GDH lost allosteric regulation by purine nucleotides. Therefore, the purpose of the antenna was deemed to likely facilitate allosteric regulation. The current antenna-less construct, which does not include bacterial sequence, has unexpected and exciting properties as allosteric regulation v by all regulators examined is maintained. In fact, the current antenna-less construct is hypersensitive to allosteric activators ADP and leucine, but the basal activity is only about 13% that of wild-type GDH. Similarly, the characterization of the HI/HA mutants found in the antenna yielded unexpected results. It has been generalized that HI/HA- GDH hyperactivity is caused by a loss of sensitivity to GTP inhibition, but this investigation revealed that dysregulation of other allosteric regulators like hypersensitivity to activation or increased basal activity of the mutants seems to be the mechanism of hyperactivity in several mutants examined. These new insights into the antenna suggest that it plays a fundamental role not only in allosteric regulation, but in catalysis as well. Therefore, it is likely that the antenna is responsible for improving enzymatic efficiency by acting as a conduit for substrate binding energy between subunits and not solely involved in facilitating allosteric regulation.