Gly456 and Arg443 are essential for allosteric regulation of human GLUD1 glutamate dehydrogenase

Abstract

Human glutamate dehydrogenase (GDH) exists in housekeeping and brain‐specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. Although the two isoenzymes share all but 15 of their 505 amino acids, they differ markedly in their allosteric regulation characteristics. To identify the structural basis of these functional properties, we performed site‐directed mutagenesis of the GLUD1 at sites that differ from the GLUD2 gene. Mutated cDNAs were expressed in Sf21 cells using the baculovirus expression system. Results showed that replacement of Gly456 with Ala renders the GLUD1 isoenzyme markedly resistant to GTP inhibition (IC50 = 2.8 ± 0.15 μm) as compared to the wild type enzyme (IC50 = 0.19 ± 0.01 μm) and abolishes its cooperative behavior. In contrast, replacement of Arg443 with Ser caused the enzyme to be essentially inactive when assayed under baseline conditions (without allosteric activators). Also, the Arg443Ser mutant was markedly resistant to ADP activation (SC50 = 345.83 ± 16.35 μm) as compared to the wild type GDH (SC50 = 24.69 ± 3.84 μm). Three other mutants (Ser331Thr; Arg470His; Asn498Ser) showed kinetic and allosteric properties similar to those of the normal GLUD1 enzyme. Hence, change of Gly456 with Ala and Arg443 with Ser accounts for some of the functional characteristics that distinguish the nerve tissue specific (GLUD2) from the housekeeping (GLUD1) GDH. Since GLUD2 has evolved from the GLUD1 gene, these data identify some of the structural changes that permitted the GLUD2 GDH to adapt to the unique metabolic requirements of the nerve tissue.