Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms: Implications for biologic function

Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific iso forms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evide nce that GDH function in vivo is regulated, and that regulatory mutations o f human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNA s in Sf9 cells and studied with respect to its regulation by endogenous all osteric effecters, such as purine nucleotides and branched chain amino acid s. Results showed that L-leucine, at 1.0 mM, enhanced the activity of the n erve tissue-specific (GLUD2-derived) enzyme by similar to 1,600% and that o f the GLUD1-derived GDH by similar to 75%. Concentrations of L-leucine simi lar to those present in human tissues (similar to 0.1 mM) had little effect on either isoenzyme. However, the presence of ADP (10-50 mu M) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation a t physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC50 = 0.20 mu M), whereas nonacti vated GLUD2 GDH was totally insensitive to this compound (IC50 > 5,000 mu M ). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the c ooperativity curves of the two isoenzymes. Kinetic studies revealed signifi cant differences in the K-m values obtained for or-ketoglutarate and glutam ate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activator s differentially altering these values. Hence, the activity of the two huma n GDH is regulated by distinct allosteric mechanisms, and these findings ma y have implications for the biologic functions of these isoenzymes.