Although it can be done to crystallize the GDH in the presence and absence of abortive complexes (NAD(P)H + GLU or NAD(P)+ + 2-oxoglutarate), crystals of apo-GDH are more fragile and diffract to lower resolution. 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. Glutamate dehydrogenase (GDH)2 is found in all living organisms and catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)+ as coenzyme (1). In eukaryotic organisms, GDH resides within the inner mitochondrial matrix where it catabolizes glutamate to feed 2-oxoglutarate to the Krebs cycle. Although there is some debate as to the directionality of the reaction, the high for ammonium in the reductive amination reaction seems to prohibit the reverse reaction under normal conditions in most organisms (2). GDH from animals, but not other kingdoms (3), is allosterically regulated by a wide array of ligands (3C9). GTP (9C11), and with 100-fold lower affinity, ATP (3), is a potent inhibitor of the reaction and acts by increasing the binding affinity for the product, thereby slowing down enzymatic turnover (11). Hydrophobic compounds such as palmitoyl-CoA (12), steroid hormones (13), and steroid hormone analogs such as diethylstilbestrol (5) are also potent inhibitors. ADP is an activator of GDH (3, 6, 10, 11, 14) that acts in an opposite manner to GTP by facilitating product release. Leucine is a poor substrate for GDH but is also an allosteric activator for the enzyme (8). Its activation is akin to ADP but acts at site distinct from ADP (15). The crystal structures of the bacterial (16C18) and animal forms (19, 20) of GDH have shown that CD38 the general architecture and the locations of the catalytically important residues have remained unchanged throughout evolution. The structure of GDH (Fig. 1) is essentially two trimers of subunits stacked directly on top Gamitrinib TPP of each other with each subunit being composed of at least three domains (19C22). The bottom domain makes extensive contacts with a subunit from the other trimer. Resting on top of this domain is the NAD binding domain that has the conserved nucleotide binding motif. Animal GDH has a long protrusion, antenna, rising above the NAD binding domain that is not found in bacteria, plants, Gamitrinib TPP fungi, and the vast majority of protists. The antenna from each subunit lies immediately behind the adjacent, counterclockwise neighbor within the trimer. Because these intertwined antennae are only found in the forms of GDH that are allosterically regulated by numerous ligands, it is reasonable to speculate that it plays a role in regulation. Open in a separate window FIGURE 1. Conformational transitions and locations of ligand binding sites in bovine glutamate dehydrogenase. of Gamitrinib TPP apo-bovine glutamate dehydrogenase with each of the identical subunits represented Gamitrinib TPP by of the shows the structure of ADP (shows the location of the inhibitor, GTP (notes the approximate location of one of the two sites (Lys-420) modified by 5-FSBA (48). Comparing and importance of GDH in glucose homeostasis was demonstrated by the discovery that a genetic hypoglycemic disorder, the hyperinsulinemia/hyperammonemia (HHS) syndrome, is caused by loss of GTP regulation of GDH (25C27). Children with HHS have increased -cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals (28). This is likely due to uncontrolled catabolism of amino acids yielding high ATP levels that stimulate insulin secretion as well.