Novel inhibitors complexed with glutamate dehydrogenase: allosteric regulation by control of protein dynamics

Abstract

Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)(+) as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

Figures

FIGURE 1.

Conformational transitions and locations of ligand binding sites in bovine glutamate dehydrogenase. A, a ribbon diagram of apo-bovine glutamate dehydrogenase with each of the identical subunits represented by different colors. The subunit arrangement is that of a trimer of dimers where anti-parallel β-strands form extensive interactions between the subunits stacked on top of each other. This pairing is represented by different shades of the same color. The conformational changes that during substrate binding are shown by the numbered arrows. As substrate binds, the NAD+ binding domain closes (1). The ascending helix of the antenna moves toward the pivot helix of the adjacent subunit (2). The short helix of the descending strand of the antenna becomes extended and distorted at the carboxyl end (3). Finally, the internal cavity of the helix compresses, bringing the three pairs closer together (4). B shows the structure of ADP (green spheres) bound to the apo-form of GDH and the location of Arg-463 (mauve spheres) that is involved in ADP activation (22). C shows the location of the inhibitor, GTP (mauve spheres), bound to the NADH (gray spheres), and glutamate (orange spheres) abortive complex. The green arrow notes the approximate location of one of the two sites (Lys-420) modified by 5′-FSBA (48). Comparing B and C, the closing of the catalytic cleft and the movement of the pivot helix is evident.

FIGURE 2.

Link between GDH and insulin homeostasis. This figure shows the role of GDH in BCH stimulated insulin secretion and how GDH inhibitors affect this process (29, 30). In energy-depleted β-cells, a BCH ramp stimulates insulin secretion. Here, the major energy source is glutaminolysis via phosphate-dependent glutaminase and GDH, because the concentration of GDH inhibitors (ATP/GTP) have been depleted and the phosphate-dependent glutaminase activator Pi (inorganic phosphate) has been increased. BCH stimulates glutamine utilization via GDH activation, thus providing the ATP signal necessary for insulin secretion. GDH inhibitors block this process by inhibiting GDH activity.

FIGURE 3.

Steady-state kinetic analysis of bovine GDH inhibition by these compounds. Shown at the top are the chemical structures of the three compounds used in these structural studies and previously identified in high throughput screens (32). The effects of hexachlorophene (HCP) on the oxidative deamination reaction has already been demonstrated (32). Shown in A and C are the steady-state velocities at varied glutamate and bithionol (Bith) or GW5074 concentrations. The marked downward trend in the curve at concentrations above 16 mm glutamate is due to substrate inhibition, and therefore the data were analyzed using the modified Monod equation (Equation 1) and summarized in Table 1. Shown in B and D are the Lineweaver-Burk plots of the oxidative deamination reaction at varying NAD+ and drug concentrations. Here, there is a marked break from the expected linearity at ∼0.1 mm NAD+ due to negative cooperativity (40). The Vmax and Km were estimated from the data at high NAD+ concentrations and summarized in Table 1.

FIGURE 4.

The structure of HCP bound to the bovine GDH·NADP+·GLU·GTP complex. A, stereo ribbon diagram of bovine GDH with the subunits colored in a manner similar to Fig. 1. For clarity, the other bound ligands (GTP, NADPH, and glutamate) are not shown in the diagram. In the central core, each of the six HCP molecules is represented by different colored spheres. B, stereo view is of the central core region looking down from the top of the hexamer, through the 3-fold axis running through the antenna. The six copies of HCP are represented by ball-and-stick models, and their corresponding electron densities are shown at a contour level of 1σ.

FIGURE 5.

The binding environment of HCP in the GDH core. A, ribbon and stick figure of the GDH·HCP complex with some of the contact residues highlighted. Note there are, in total, three molecules of HCP bound in conformation A and three in conformation B. The orientation is with the 3-fold axis running vertically through the middle of each panel. The color of the surfaces corresponds to the subunit colors used in Fig. 3. B, a stereo image using a similar view orientation and shows a stereo image of a surface rendering of the internal core of GDH with three of the six bound HCP molecules represented by ball-and-stick models.

FIGURE 6.

Electron density and binding environment of the bound bithionol. The view and coloring are the same as the top of Fig. 3, looking toward the core of the enzyme along one of the 2-fold axes and perpendicular to the 3-fold axis. A, a stereo image of the bithionol·GDH complex with the drug molecules represented by ball-and-stick models and contact residues are noted. B, a stereo view of the atomic surface of the binding site, colored according as per all of the other figures. Note that this cavity is visible from the exterior of the hexamer.

FIGURE 7.

Electron density and binding environment of the bound GW5074. A, stereo image with essentially the same view as used for bithionol in Fig. 5 with the electron density. B, the molecular surface of the binding cavity. This binding site is the same as bithionol.

FIGURE 8.

Locations of the all of the bound ligands. The purpose of this figure is to elucidate the locations of all of the ligand binding sites on bovine GDH. A, a ribbon diagram showing the structures of bithionol (spheres with various colors), NADPH (gray spheres), glutamate (yellow spheres), GTP (black spheres) bound to GDH. The view is the same as in Fig. 5 and shows that the bithionol (and GW5074) molecules bind right around the 2-fold axes relating the pairs of GDH subunits. B, stereo figure showing a cutaway of the core region with the locations of bithionol and HCP denoted by the colored spheres. The view here is down the 3-fold axis with the three 2-fold axes lying in the plane of the figure and running between the bithionol pairs. Note that the HCP molecules cluster in the core of the enzyme, and bithionol and GW5074 bind more toward the exterior of the hexamer.

FIGURE 9.

Conformational differences between the open and closed forms of the enzyme. In this stereo diagram, the C-α backbones of the subunits in the closed form are colored in darker hues while the subunits of the aligned open form of the enzyme are colored in corresponding lighter hues. The glutamate binding domain of the red (closed form) subunit was aligned to that of the pink (open form) subunit. Because the stacked dimers of subunits act as rigid bodies, this also aligns the dark blue (closed form) subunit with the light blue (open form) subunit. This area is highlighted by the yellow square. The general location of the drugs is denoted by the yellow circle. The yellow arrows highlight the helices in the core region that are involved in drug binding and spread apart when the catalytic sites open.

FIGURE 10.

Schematic of the various vectors between the six subunits of GDH. The coloring is the same as the other ribbon diagrams, and each subunit is represented by a sphere to emphasize the trimer of dimer configuration. There are four unique distances in the hexamer; between subunits within a trimer (intra-trimer), between subunits within a dimer (intra-dimer), and two cross vectors between each subunit and the two opposite subunits on the other trimer (cross-vectors 1 and 2). The distances from the various complexes are summarized in Table 3.

FIGURE 11.

Effects of the drugs on GDH from other kingdoms. Shown here is the effect of varying concentrations of the three drugs on GDH from bovine (red lines), tetrahymena (green lines), and E. coli (blue lines). Curve fitting results are summarized in Table 4.