GLDH

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Introduction

Glutamate dehydrogenase (GLDH, or GDH) is a hexameric enzyme responsible for the reversible conversion of glutamate to α-ketoglutarate and ammonia, while reducing NAD(P)⁺ to NAD(P)H. In addition to promoting Krebs cycle anaplerosis and energy production, GDH is also closely related to cell signal transduction and redox homeostasis.

Different GDH isoenzymes are usually expressed in lower life forms such as bacteria or yeasts, and these isoenzymes have strict specificity for NAD⁺ or NADP⁺. NAD⁺ dependent GDH mainly plays a metabolic role, while NADP⁺ specific enzymes are involved in biosynthetic functions. Three different GDH isozymes have been identified in the yeast Saccharomyces cerevisiae: yGDH1 (NADP⁺-specificity), yGDH2 (NAD⁺-specificity) and yGDH3 (NADP⁺-specificity). NAD⁺-specific isoenzyme (yGDH2) plays a role in the oxidative deamination of glutamate to α-ketoglutarate and ammonia, and NADP⁺-specific isoenzymes (yGDH1, yGDH3) are mainly involved in glutamate biosynthesis. In lower organisms, the regulation of GDH is achieved at the transcriptional level. In Arabidopsis thaliana, NAD⁺ specific GDH is encoded by three different genes (aGDH1, aGDH2 and aGDH3). In Oryza sativa (rice), NAD⁺ specific oGDH is also encoded by three different genes which show different expression patterns. At the same time, NADP⁺ specific GDH proteins have also been identified in a series of higher plants. In mammals, GDH has developed into a highly regulated enzyme with dual coenzyme specificity. Mammalian GDH1 (hGDH1 in humans) can play an important role in catabolism and cell synthesis. In addition, GDH1 is also controlled by a complex allosteric regulation system.

Figure 1. The glutamate dehydrogenase (GDH) pathway and the Krebs cycle function (Plaitakis, A.; et al. 2017)

Structure

GDH is a hexamer composed of six subunits. Each of these peptides contains approximately 500 amino acids and has a molecular weight of approximately 56 kDa. The hexamer enzyme is composed of two trimers, each of which contains three functional domains: NAD⁺ binding, glutamate binding and regulatory domains. In the trimer, the antennas of the adjacent subunits are intertwined with this inter-subunit interaction thought to mediate allostery, but the exact mechanism is not fully understood.

In recent decades, the acquisition of high-resolution X-ray structures of different GDHs has provided us with an in-depth understanding of the molecular mechanisms of their catalytic functions. Recent studies on mammalian GDH1 (the advanced structure including the "antenna") have shown that the hexamer structure undergoes significant conformational changes in each catalytic cycle. It has been observed that when the catalytic cleft opens, the NAD⁺ domain moves away from the glutamate binding domain and rotates around the antenna in a clockwise direction. Those observations indicate that mutations of amino acids located in this helix in hGDH1 weaken the GTP inhibitory effect and cause hyperinsulinemia/hyperammonemia (HI/HA) syndrome, thus emphasizing the importance of the small helix.

Figure 2. Structural model of hGDH1 (Plaitakis, A.; et al. 2017)

The GDH Catalysis

Studies have shown that the thermodynamic equilibrium of mammalian GDH can promote the synthesis of glutamate, but it is still unclear whether the enzyme acts to the direction of oxidation-deamination in vivo. The catalytic effect of GDH is affected by substrate concentration, ionic strength, pH, and buffer composition. Studies on purified GDH from mammalian tissues or recombinant hGDH1 show that the optimal pH range of the enzyme is 7.75-8.00, and ADP activation is significantly reduced at lower pH values. The optimal pH of hGDH2 is 7.50, and the enzyme can also operate effectively at even lower pH values (7.25 to 7.0). Due to the relatively high Km of ammonia, it is expected that GDH will operate to the direction of oxidative deamination, especially in tissues where the ammonia concentration is usually low.

Site-directed mutagenesis in hGDH1 at sites that differ in hGDH2 showed that a single evolutionary amino acid substitution (Arg443Ser) located in the antenna's descending chain reduced catalytic activity and eliminated L-leucine activation. In addition, the changes in Arg443Ser make the enzyme heat-labile and increase its sensitivity to steroid hormones. Other functional analysis of human GDHs shows that the basic activity of hGDH2 depends on the concentration of recombinase during the assay. The dependence of the wild-type hGDH2 and hGDH1 mutant on enzyme protein concentration is thought to be related to the disruption of the Arg443-Ser409 hydrogen bond in the antennae. Unlike the case of hGDH2 and hGDH1 mutants, changing the protein concentration of wild-type hGDH1 has little effect on its specific activity.