The structure and allosteric regulation of glutamate dehydrogenase

Abstract

Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a 'housekeeping' enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an 'antenna' like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy.

Figure 1

Atomic structure of animal glutamate dehydrogenase. This is a ribbon diagram of bovine glutamate dehydrogenase complexed with glutamate (yellow), NADH, and the inhibitor GTP (brown). There are two molecules of NADH bound; one at the active site (grey) and one at the allosteric inhibitory site (cyan). ADP also binds at this second NADH site and activates the enzyme. The ribbons are colored according to three pairs of dimers with shades of blue, red, and green.

Figure 2

Atomic details of the ADP and GTP allosteric regulatory sites on animal GDH. In these figures, the residue numbering of human GDH is used. The side chains that form hydrogen bonds with the ligands are represented by sticks and colored according to atom type.

Figure 3

Overview of the multi-organ effects of HHS. The top figure shows that the loss of GTP inhibition increases the flux of glutamate through GDH. The resulting stimulation of Krebs cycle activity results in higher a ATP:ADP ratio and causes degranulation of the β-cells. The middle figure shows that in the liver and/or the kidneys, the increased activity of GDH diminishes the glutamate pool that results in lower N-acetylglutamate levels followed by a loss of carbamoyl phosphate synthetase activation. This leads to higher serum ammonium levels not only due to the deamination of glutamate but also the decrease in urea synthesis. The bottom panel shows the large number of glutamate dependent ion channels involved in neuronal synapsis and the role that glial cells play in glutamate recycling. Unregulated GDH will very likely affect glutamate homeostasis.

Figure 4

Locations of the HHS mutations. Shown here is a ribbon diagram of one GDH subunit. The side chains of the known HHS mutations are represented as stick figures colored according to the atom type. The numbering used here correlates to the human GDH amino acid sequence. The structure of bound GTP is shown as a brown stick figure. The residues with underlined labels are those that are not in contact with the bound GTP and are clustered around the antenna region.

Figure 5

Structures of some of the new and novel inhibitors for GDH. The compounds fall into three general classes; the tea polyphenols, large soluble compounds, and small hydrophobic molecules. The three polyphenols highlighted in yellow do not inhibit GDH activity even at high concentrations even though they have essentially the same chemical properties.

Figure 6

Locations of the binding sites for the small hydrophobic compounds. On the left, the orange and mauve molecules at the GDH two-fold axes represent the pair of drugs bound at the expansion point between the dimers of GDH subunits. The figure on the right is a top-down view of the core of the enzyme showing the relative locations of bithionol and hexachlorophene. Hexachlorophene also binds as three pairs of molecules that are represented by cyan and black molecules.

Figure 7

Effects of polyphenols on GDH. A) Using steady-state kinetic assays on purified enzyme, this figure shows that only EGCG and ECG inhibit the enzyme with nanomolar ED₅₀’s while the chemically similar compounds, EC and EGC, had no effect on GDH activity. B) This figure shows that EGCG inhibits five different GDH mutants that lead to the HHS syndrome. Also shown here is the fact that Tetrahymena GDH, that has an antenna and activated by ADP, is also affected by EGCG. However, if the antenna is removed from human GDH and replaced by the short loop found in bacterial sources, EGCG has no effect on the enzyme. C) Using β-cell islet perifusion assays, EGCG clearly blocks GDH-mediated, BCH stimulation of insulin secretion in a dose-dependent manner. Importantly, EGC, that was shown to be in active in figure (B), has no effect on insulin secretion.