| Abstract |
Mammalian glutamate dehydrogenase (GDH) has been shown to play an important role in cellular homeostasis by interconnecting amino acid and carbohydrate metabolism. In mammalian brain, where GDH is localized mainly in astrocytes, there is evidence that this enzyme contributes to the metabolism of the neurotransmitter glutamate. Human GDH exists in two isoforms, which are encoded by the GLUD1 and GLUD2 genes, respectively, and share, in their mature form, all but 15 of their 505 amino acids. The GLUD1 gene is autosomal, contains introns and is expressed in all tissues, including brain tissue. On the other hand, the intronless GLUD2 gene is X-linked and is expressed only in brain and testis. The two human GDH isoenzymes differ significantly in their kinetic properties and their allosteric regulation, despite the small differences in their amino acid sequence. Specifically, GLUD1 is inhibited potently by GTP and this inhibition shows positive cooperativity, while GLUD2 GDH requires significantly higher GTP concentrations to be inhibited and this inhibition is characterized by negative cooperativity. Moreover, the GLUD2 enzyme in the absence of ADP or L-leucine is essentially inactive, in contrast to the GLUD1 isoenzyme, which shows a significant basal activity in the absence of activators. Addition of ADP or L-leucine activates the GLUD2 isoenzyme, restoring its activity to levels comparable to those of GLUD1 GDH. The above properties probably permit the GLUD2 isoenzyme to function under the unique conditions of the nervous tissue, such as the states of low energy charge (high ADP:ATP ratio) occurring during intense glutamatergic transmission and the high GTP concentrations that are found in brain. Aiming to decipher the structural basis of these significant functional differences between the two GDH isoenzymes, we performed site-directed mutagenesis of the GLUD1 enzyme at sites that it differs from the GLUD2 isoenzyme, replacing each amino acid at these sites with the corresponding amino acid of the GLUD2 GDH. Our studies showed that replacement of Gly456 by Ala made the enzyme resistant to GTP inhibition and led to the loss of the positive cooperativity associated with this inhibition. Moreover, replacement of Arg443 by Ser abolished the basal activity of the GLUD1 enzyme and rendered the enzyme dependent on ADP for its function. Mutagenesis at other sites that viii differ between the two GDH isoenzymes (Ser331Thr, Met370Leu, Met415Leu, Arg470His και Asn498Ser) did not affect the allosteric regulation and the kinetic properties of the GLUD1 isoenzyme. The above results showed that the main functional differences between the two human GDH enzymes can be attributed to only two amino acid changes (Arg443Ser and Gly456Ala). It has been shown that the intronless GLUD2 gene has evolved from the GLUD1 gene by retrotransposition in a common ancestor of man and modern apes. Consequently, the combination of random mutations and the pressure of natural selection gave the GLUD2 gene its present properties. The two amino acid substitutions Arg443Ser and Gly456Ala are, according to our studies, the main evolutionary changes that led to the adaptation of GLUD2 GDH to the unique metabolic needs of the nervous tissue, possibly contributing to the significant functional differentiation of the human brain as compared to that of other mammals.
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Μελέτη των δομικών και λειτουργικών διαφορών μεταξύ των δύο ανθρώπινων ισοενζύμων γλουταμικής αφυδρογόνασης
