Perhaps one of the most confusing regulatory sites on mammalian GDH is the allosteric activator, ADP, binding site (). The existence of a second NADH binding site per subunit was demonstrated both kinetically and by binding analysis [25
]. It was observed that NADH alone binds with a stoichiometry of 7–8 molecules per hexamer. In the presence of glutamate, NADH binds more tightly and the stoichiometry increases to 12 per hexamer [27
]. Similarly, GTP also increases the affinity and binding stoichiometry [6
]. This second coenzyme site strongly favors NADH over NADPH with Kd’s of 57μM and 700μM, respectively. In the case of oxidized coenzyme, NAD+
, two binding sites were also observed. While the recent structures of the various complexes have demonstrated that ADP and NAD(H) bind to the same site [9
], this was first suggested by ADP binding competition with NAD+
] and NADH [7
]. Further, these binding studies provided direct evidence that GTP and glutamate enhance binding of NADH to a second site and ADP blocks binding of both NAD+
and NADH to a second site. Since, in general terms, NADPH is involved in anabolic reactions in the cell while NADH is important for catabolic processes, it is possible that this regulation offers a feedback mechanism to curtail glutamate oxidation when catabolic reductive potentials (NADH) are high.
In nearly every way, ADP acts in a manner opposite to NADH binding to this site. In the oxidative deamination reaction, ADP activates at high pH, but inhibits at low pH with either NAD+
as coenzyme. In the reductive amination reaction, ADP is a potent activator at low pH and low substrate concentration. At pH 6.0, high concentrations of α-KG and NADH, but not NADPH, inhibit the reaction. This substrate inhibition is alleviated by ADP [4
]. Therefore, while GTP and glutamate bind synergistically with NADH to inhibit GDH, ADP activates the reaction by decreasing the affinity of the enzyme for coenzyme at the active site. Under conditions where substrate inhibition occurs, this activates the enzyme. However under conditions where the enzyme is not saturated (e.g. low substrate concentrations), this loss in binding affinity causes inhibition. Put another way, under conditions where product release is the rate-limiting step, ADP greatly facilitates the catalytic turnover. It should be noted that the fact that substrate (2-oxoglutarate) inhibition in the reductive amination reaction is only observed using NADH as coenzyme was suggested to be due to NADH (but not NADPH) binding to the second coenzyme site. Further, it was suggested that ADP activation under these conditions was due to ADP displacement of NADH from the second allosteric site [2
ADP binds behind the NAD binding domain and immediately under the pivot helix [9
]. As shown in , R459 lies on the pivot helix and interacts with the phosphates of the bound ADP. It was proposed that this interaction might facilitate the rotation of the NAD binding domain and the release of product [9
]. To test this, R459 (R463 in human GDH) was mutated to an alanine and this led to a loss in ADP activation. This essentially suggests that ADP activates the reaction by ‘pulling’ on the back of the NAD binding domain to help open the active site cleft and facilitating product release. More precisely, ADP likely decreases the energy required to open the catalytic cleft.
To better understand how ADP can activate GDH and to put allosteric regulation in broader context, it is helpful to review the two main models for explaining homotrophic allosteric regulation; the concerted and sequential models. The concerted, or symmetrical, model was first proposed by Monod, Wyman, and Changeux [29
]. In this model (MWC), all subunits in an oligomer have the same structure but that they can exist in two different states; relaxed (R) and tense (T) where the relaxed state binds ligand tighter than the tense state. In enzymes exhibiting positive cooperativity, the structural equilibrium of the whole oligomer shifts towards the R state and the affinity of the substrate to the enzyme increases as the substrate binds to the active site. While this model is easily applied to systems exhibiting positive cooperativity, it fails to describe negative cooperativity. The concerted model would require substrate to preferentially bind to the low affinity species (T state) in order to decrease affinity with increasing saturation.
To explain how negative cooperativity might occur, Koshland, Nemethy, and Filmer developed the sequential model that is applicable to both negatively and positively cooperative enzymes [30
]. The main difference between the two models is that the sequential model does not require all of the subunits in the oligomer to adopt the same conformation. Instead, substrate binding to a subunit can induce a particular conformation and this change can affect other subunits in the oligomer by making it either thermodynamically harder or easier for them to make the same transition. These transitions become easier as the enzyme becomes saturated in the case of positive cooperativity and are harder with negative cooperativity. In the case of GDH, that exhibits negative cooperativity with respect to coenzyme binding, substrate binding to the first subunit may cause the catalytic cleft to close and, because of interactions at the subunit interfaces, this conformation transition makes it harder (negative cooperativity) for the adjacent subunits to clamp down upon substrates as they bind. Therefore, the sequential model is attractive since it can be applied to both positively and negatively cooperative systems but also allows for more independent and fluid structures among the subunits in the oligomer. From the structures of GDH, we are not seeing obvious ‘R’ and ‘T’ states of the enzyme and the MWC model seems overly simplistic even for heterotrophic allosteric regulation. Indeed, the subunits all have slightly different conformations in the apo form of GDH. Instead, it seems more likely that all of the conformational changes that occur at subunit interfaces, that are required for for catalytic turnover, are likely facilitated by activating ligands such as ADP while inhibited by inhibitors such as GTP.
The structures of GDH complexed with NADH, NADPH, and NAD have all been determined [18
]. Because NADH (but not NADPH) has been suggested to be an inhibitor of the reaction, it is somewhat surprising that it binds to the ADP activation site [9
]. The adenosine-ribose moiety location exactly matched that of ADP. The electron density of the ribose-nicotinamide moiety was much weaker and was initially built in two alternative conformations. However, the stronger density for this portion of NADH suggests that it points down into the interface between adjacent subunits. As predicted from the binding studies reviewed above, NADPH was found bound to the active site but not the second, allosteric site.
was found to bind in a manner essentially identical to NADH [18
]. From steady state kinetic analysis, it was initially thought that NAD+
binding to this second site causes activation of the enzyme [25
], even though NADH causes apparent inhibition. However, subsequent studies demonstrated that this apparent activation was due to negatively cooperative binding with respect to coenzyme [32
]. Therefore, it is not clear what difference there might be, if any, between NAD+
and NADH binding to GDH at this location. It is interesting to note that modification of the ADP site with an ADP analog did not eliminate NADH inhibition [33
]. Perhaps this is due to the nicotinamide moiety still binding to the pocket between the subunits in spite of AMPSBDB being bound to R459. As will be detailed below, recent studies on new GDH inhibitors have shown that compounds binding to subunit interfaces, and at the ADP site itself, can be potent inhibitors of the enzyme. Perhaps the ribose-nicotinamide moiety is acting in a similar manner.
The physiological role of ADP activation is easily understood; when the energy level of the mitochondria is low and ADP levels are high, the catabolism of glutamate is facilitated for energy production. However, the possible in-vivo
role of NADH inhibition is less clear. Since NADH inhibition is observed at concentrations above 0.2mM (e.g. see [34
]), but only reaches ~50% inhibition at 1mM NADH, NADH inhibition seems more likely to work synergistically with GTP regulation; under conditions of high reductive potential, NADH acts with GTP to keep GDH in a tonic state.
At an atomic level, there is a very clear delineation between ligands binding to the open and closed conformations. NADH alone only binds to the active site. When glutamate is added, the catalytic cleft closes and NADH is able to bind to the second, allosteric site. Further, the GTP binding site collapses when the catalytic cleft opens and therefore GTP also favors the closed conformation. Therefore, the synergism between NADH and GTP is likely due to both ligands binding to, and stabilizing the closed conformation. Again, this supports the contention that NADH inhibition alone may not have a significant physiological role, but rather its main function is the enhancement of GTP inhibition.