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Untangling the glutamate dehydrogenase allosteric nightmare
Review
Untangling the glutamate dehydrogenase allosteric nightmare Thomas J. Smith1 and Charles A. Stanley2 1 2
Donald Danforth Plant Science Center, 975 North Warson Road, Saint Louis, MO 63132, USA Endocrinology Division, Abramson Research Center, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the ‘missing link’ in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion. Fifty years of GDH studies Glutamate dehydrogenase (GDH) is found in all living organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NADP+ as the coenzyme [1]. This homohexameric enzyme has subunits comprising 450 and 500 amino acids in bacteria and animals, respectively. 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 Michaelis constant (Km) for ammonium in the reductive amination reaction seems to prohibit the reverse reaction under normal conditions in most organisms [2]. Even in plants, recent 15N-incorporation studies in the presence of excess ammonium have shown that GDH functions in the oxidative deamination reaction [3]. However, some bacteria use GDH rather than the normal glutamine synthetase–glutamate synthase pathway to fix nitrogen under high ammonia conditions [4]. The work reviewed here lends extremely strong support for GDH mainly operating in the oxidative deamination reaction in animals. Under most in vitro conditions, coenzyme release is the rate-limiting step, particularly at higher substrate concentrations [5]. From the properties of the most important allosteric regulators noted later, it seems highly likely that product release is the rate-limiting step in vivo as well. The crystal structures of the bacterial [6–8] and animal forms [9,10] of GDH have shown that the locations of the catalytically important residues have remained Corresponding author: Smith, T.J. ([email protected])
unchanged throughout epochs of evolution. However, GDH from animals, but not other kingdoms [11], is allosterically regulated by a wide array of ligands [11–17] (Table 1). GTP [17–19] (and, with 100-fold lower affinity, ATP [11]), is a potent inhibitor of the reaction and functions by increasing the binding affinity for the product, thereby slowing down enzymatic turnover [19]. Hydrophobic compounds such as palmitoyl coenzyme A (CoA) [20], steroid hormones [21] and steroid hormone analogs such as diethylstilbestrol (DES) [13] are also potent inhibitors. ADP is an activator of GDH [11,14,18,19,22], which functions 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 [16]. Its activation is akin to ADP but it acts at a site distinct from ADP [23]. Mammalian GDH exhibits several unusual kinetic properties with unclear physiological roles. Negative cooperativity is observed as nonlinear Lineweaver-Burk plots when NADP+ is varied [24]. Subsequent studies demonstrated that coenzyme (NADPH) binding to the initial subunits weakens the affinity to subsequent subunits [25,26]. This process is dependent upon the substituent at the a carbon of the substrate backbone [27]. The physiological role for this behavior has been suggested to maintain a particular catalytic rate or responsiveness as coenzyme concentrations vary in the mitochondria [28]. However, it is also possible that negative cooperativity is a consequence of inter-subunit communication necessary for other purposes. This communication has been shown by abrupt and striking changes in the circular dichroism and fluorescence spectra when GDH is half saturated [26] and from chemical modification studies demonstrating that the loss of enzymatic activity is disproportional to the number of subunits modified [29–31]. There is marked substrate inhibition in both reaction directions that is exacerbated by GTP and antagonized by ADP [14]. This might be more indicative of product release being the rate-limiting step rather than a form of regulation per se. GDH also has a second binding site for NADH that has been suggested to inhibit the enzyme and bind synergistically with GTP [12,32,33]. However, this inhibition only occurs at high, non-physiological concentrations. Animal GDH has been extensively studied since the early 1950 s. However, the mechanisms of allostery and the reason why animals created such a tangled network of regulation has been unclear until very recently. Structural studies have started to deconvolute mammalian GDH allostery, its possible evolutionary origins and, from
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Table 1. Summary of GDH regulation Type of regulation Regulator GTP Inhibition Palmitoyl CoA NADH (second site) ATP Diethylstilbestrol (DES) Zn2+ Leucine Activation BCH ADP Negative cooperativity Substrate inhibition
Coenzyme Glutamate 2-oxoglutarate
Behavior Increases coenzyme affinity and stabilizes abortive complex. Antagonist with ADP. Longer fatty acids are more effective than shorter chains. Inhibits at high concentrations. Agonist with GTP. Similar to, but 100 times weaker efficacy than, GTP. Inhibition reversed by ADP and leucine. Reversed by ADP and stabilizes abortive complex. Also a substrate. Activation differs to ADP but also an antagonist to DES and GTP. Non-hydrolysable form of leucine that also activates the enzyme. Decreases affinity for coenzyme and abortive complex. Antagonist for GTP. Can inhibit at low coenzyme concentrations. Observed in binding studies for all forms, observed kinetically for NADP+. Second position substituent on glutamate backbone is essential. Strongest at higher pH. Strongest at lower pH.
analysis of a severe hyperinsulinemic hypoglycemia disorder, demonstrate the importance of GDH allosteric regulation in humans. The structure of mammalian GDH The structure of mammalian GDH (Figure 1) is two trimers of subunits stacked directly on top of each other with each subunit being composed of at least three domains [9,10,34,35]. 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. Rising above these two domains is a long protrusion, ‘antenna’, that is a helix– loop–helix composed of 50 residues, which is not found in bacteria, plants, fungi and the vast majority of protists. The antenna from each subunit lies immediately behind the adjacent, counter-clockwise neighbor within the trimer. That these intertwined antennae are only found in the forms of GDH allosterically regulated by numerous ligands leads to the obvious conclusion that it plays a major part in this regulation. By mixing the coenzyme from one side of the reaction with a substrate from the other side of the reaction, it is possible to saturate GDH with ‘abortive’ or ‘dead-end’ complexes. When compared with the apo form of the enzyme, it is clear that GDH performs a complex ballet during catalytic turnover [9,10,34,35] (Figure 1). The substrate binds to the deep recesses of the cleft between the NAD-binding domain and the lower domain. The coenzyme binds along the NAD-binding-domain surface of the cleft. Upon binding, the NAD-binding domain rotates by 188 to firmly close down upon the substrate and coenzyme (Figure 1; arrow 1). As the catalytic cleft closes, the base of each of the long ascending helices in the antenna seems to rotate out in a counter-clockwise manner to push against the ‘pivot’ helix of the adjacent subunit (Figure 1; arrow 2). There is a short helix in the descending loop of the antenna that becomes distended and shorter as the mouth closes in a manner akin to an extending spring (Figure 1; arrow 3). The pivot helix rotates in a counter clockwise manner along the helical axes in addition to rotating counter clockwise around the trimer threefold axis. Finally, the entire hexamer seems to ‘exhale’ as the mouth closes (Figure 1; arrow 4). The three pairs of subunits that sit on top of each other move as rigid units towards each other, compressing the cavity at the core of the hexamer. 558
Refs [17–19] [20] [12,32,33] [11] [20] [61] [16] [15,62] [11,14,18,19,22] [24–26] [14] [14]
Structural mechanisms of allosteric regulation From the structures of various GDH–ligand complexes (Figure 1), GDH allosteric regulation is likely to be correlated with these complex motions. GTP binds to the side of the ‘jaw’ of each subunit (Figure 1c). As the NAD-binding domain closes, a space is opened between the pivot helix and the back of the domain. This affords enough space for GTP to bind mainly via interactions with its triphosphate moiety [9,34]. This explains why GTP increases the affinity for coenzyme; product release is made more difficult. By contrast, ADP binds behind the NAD-binding domain (Figure 1b) immediately under the pivot helix [35]. ADP cannot activate by locking the catalytic mouth open because that would also prevent catalytic turnover. Instead, it was proposed that ADP activates by helping the mouth to open by interacting via a basic residue on the pivot helix (Arg463) [35]. Indeed, if Arg463 is mutated to alanine, the enzyme is still able to bind ADP but is no longer activated by it [35]. This gives a physical explanation for the ADP and GTP antagonism in that they have opposite effects on the motion of the NAD-binding domain and also indirectly affect each other’s binding sites. At the physiological level, these two ligands are acting as energy sensors. When the mitochondria are at a high energy state and rich in triphosphates, GDH is inhibited. When the mitochondria are low in energy, the elevated ADP levels activate GDH to catabolize glutamate and feed the Krebs cycle with 2-oxoglutarate. The ADP and GTP antagonism indicates that GDH is kept in a tense state in which activity is finely tuned depending on subtle changes in the ADP:GTP ratios. The complex series of motion associated with catalytic turnover might also lend some clues as to how negative cooperativity is exacted. Because the ADP and apo forms of the enzyme are in the ‘open’ state, it seems safe to assume that this conformation represents the lowest energy. Therefore, perhaps increasing amounts of energy are required to saturate the enzyme as the enzyme is forced into the more compressed protein conformation. In the case of animal GDH, this unfavorable energy probably comes from a combination of the changes in the antenna, the position of the NAD-binding domain or the compression of the core. An exciting prospect is that perhaps some, or all, of these changes suddenly ‘snap’ into place as the enzyme becomes saturated, as indicated by the previous spectroscopy experiments [26]. A larger question is what is
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Figure 1. Structure and regulation of GDH. (a) The apo form of bovine GDH with each of the identical subunits shown in different colors. The light and dark hues show the dimeric pairing of the subunits, and these dimers move in and out as the catalytic mouth closes and opens. The arrows show regions of motion during catalytic turnover. As the NAD-binding domain closes on the substrate and coenzyme (arrow 1), the antenna from the adjacent subunit pushes against the pivot helix (arrow 2), a helix in the descending strand of the descending strand of the antenna becomes distorted (arrow 3) and the core of the enzyme compresses (arrow 4). (b) A close up of the ADP (green spheres) complex. Also shown is the side-chain of Arg463 (mauve) that, when mutated to an alanine, abrogates ADP activation. (c) Bovine GDH complexed with NADPH (gray), glutamate (orange) and GTP (mauve).
the advantage of negative cooperativity when the enzyme could have been made as a single subunit or with the subunits in more isolated environments? One possibility is that negative cooperativity might make the enzyme more efficient by transferring the energy associated with substrate binding to one subunit to facilitate product release from other subunits [36]. This is akin to a car engine where the explosion in one cylinder compresses the gas/air mixture in another. Because some of these motions have been observed in the more primitive forms of GDH [7,37], such conservation of binding energy could have been part of the original evolutionary design of this
enzyme. The following discussion focuses on the mechanisms and evolution of regulation by ligands binding to sites other than the active site (hence the Greek word allostery; allos ‘other’, stereos ‘shape’) that appeared along the way to the evolution of animal GDH (Box 1). The role of animal GDH allostery Although the structures have defined the locations of many of the allosteric sites, the reason for the sudden appearance of regulation in animals was not at all clear. A clue to this puzzle came from studies that linked GDH regulation with insulin and ammonia homeostasis. The mitochondrion of 559
Review Box 1. Models of allosteric regulation There are two widely accepted models for the induction of allostery in oligomeric proteins, the Monod-Wyman-Changeux (MWC) model [59] and the Koshland-Nemethy-Filmore (KNF) model [28,60]. The MWC model describes the positive cooperative binding of oxygen to hemoglobin by indicating that the subunits are in equilibrium between a low-affinity (T) and a high-affinity (R) state. Added ligand will bind to the R state and the equilibrium will adjust by moving more of the T-state subunits to the R state. In this way, the ligand binds in a positively cooperative manner; the affinity of the ligand for the protein increases with increasing ligand concentration. However, this model cannot explain situations in which ligands bind in a negatively cooperative manner. Alternatively, the KNF model indicates that ligand binding to one subunit can induce conformational changes in the other subunits resulting in either an increase (positive cooperativity) or decrease (negative cooperativity) in ligand affinity. The results reviewed here indicate that allosteric regulation of GDH probably occurs via a mechanism most in line with the KNF model. The homotrophic allostery observed with negatively cooperative binding of coenzyme clearly is well described by the KNF model. However, the heterotrophic allostery represented by GTP and ADP regulation is not exactly as described by KNF. It is clear that the antenna is involved and, therefore, it seems likely that there is communication among the subunits. However, the structural studies do not show that these allosteric regulators induce discreet conformation changes but, rather, it seems more likely that they are affecting the amount of energy required for the domain motion required for catalysis.
the pancreatic b-cell have an integrative role in the fuel stimulation of insulin secretion (Figure 2). The current concept is that mitochondrial oxidation of substrates increases the cellular phosphate potential that is manifested by a rise in the ATP4–:MgADP2– ratio. This, in turn, closes the plasma membrane KATP channels to depolarize the membrane potential, opens voltage-gated Ca2+ channels, causes a rise of free cytoplasmic Ca2+ and leads to insulin granule exocytosis. The connection between GDH and insulin regulation was initially established using a nonmetabolizable analog of leucine [15,38], BCH (b-2-aminobicyclo[2.2.1]-heptane2-carboxylic acid). These studies demonstrated that activation of GDH was tightly correlated with increased glutaminolysis and release of insulin. In addition, it has also been noted that factors that regulate GDH also affect insulin secretion [39]. It was suggested that glutamate serves as a mitochondrial intracellular messenger when glucose is being oxidized and that the GDH participates in this process by synthesizing glutamate [40]. However, as discussed before, the hypothesis that GDH (with a very high Km for ammonium) functions in the reductive amination reaction in vivo is controversial [41]. Subsequently, it was postulated that glutamine could also have a secondary messenger role and that GDH plays a part in its regulation [42–44]. The in vivo importance of GDH in glucose homeostasis was demonstrated by the discovery that a genetic hypoglycemic disorder, the hyperinsulinemia/hyperammonemia (HI/HA) syndrome, is caused by loss of GTP regulation of GDH [45–47]. Therefore, allosteric regulation of GDH is central to understanding the response of the b-cell to the cellular fuel state. Children with HI/HA have increased b-cell responsiveness to leucine and susceptibility to hypoglycemia after highprotein meals [48]. This is likely to be due to uncontrolled 560
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catabolism of amino acids yielding high ATP levels that stimulate insulin secretion and high serum ammonium levels. The elevation of serum ammonia levels reflects the consequence of altered regulation of GDH in liver, leading to increased ammonia production from glutamate oxidation and, possibly, also impaired urea synthesis by carbamoylphosphate synthetase due to reduced formation of its activator, N-acetyl-glutamate, from glutamate. This genetic lesion disrupts the regulator linkage between glycolysis and amino acid catabolism. During glucose-stimulated insulin secretion in normal individuals, it has been proposed that the generation of high-energy phosphates inhibits GDH and promotes conversion of glutamate to glutamine, which, alone or combined, might amplify the release of insulin [42,43]. Further support for this contention came from studies on the inhibitory effects of the polyphenolic compounds (Figure 3) from green tea on BCH-stimulated insulin secretion [49]. In these studies, normal b-cells are first ‘run down’ such that they are at a low-energy state (low GTP and ATP levels). When BCH is added, GDH is stimulated to catabolize glutamate, 2-oxoglutarate is formed and fed into the Krebs cycle, ATP levels are increased and insulin is released. Of the four prevalent polyphenols found in green tea [epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epicatechin (EC) and epigallocatechin (EGC)] only EGCG and ECG inhibited both wild-type and HI/HA mutant forms of GDH activity in vitro. The notable exception was that EGCG did not inhibit GDH if the antenna had been removed. This specificity carried over into the perfused-islet studies that demonstrated that EGCG, but not EGC, blocked GDH-mediated insulin secretion. This not only lends support for the contention that GDH has an important role in insulin homeostasis but also indicates that the HI/HA disorder might be directly controlled pharmaceutically. Evolution of GDH The structure of mammalian GDH shows that the 50residue antenna represents a clear departure from its ancestral forms and is part of the concerted movement of the enzyme subunits that occurs during catalytic turnover. However, it was not initially clear when and why this feature suddenly evolved. Recent genomics work has shown that the Ciliates within the Protista have an antenna that is slightly smaller than that found in the Animalia kingdom in spite of the fact that other members of the Protista, such as trypanosomes, have GDH nearly identical to bacterial forms [50]. Therefore, the Ciliates are an unexpected evolutionary ‘missing link’ in GDH development. To try to address the reasons for the evolution of the antenna, the allosteric behavior of several forms of GDH were examined [50]. With regard to regulation, Tetrahymena GDH is, indeed, between kingdoms; it is activated by ADP and inhibited by palmitoyl CoA, but unaffected by GTP and leucine. Interestingly, the lack of leucine activation of Tetrahymena GDH indicates, but does not prove, that there could, indeed, be a secondary binding site for leucine. The catalytic site is highly conserved between the GDHs from the various sources. Therefore, if activation
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Figure 2. Link between GDH and insulin homeostasis. (a) The role of GDH in BCH-stimulated insulin secretion and the effects of EGCG on this process. In energy-depleted bcells, a BCH ramp stimulates insulin secretion. Here, the most importnant energy source is glutaminolysis via phosphate-dependent glutaminase (PDG) and GDH, because the concentration of GDH inhibitors (ATP and GTP) has been depleted and the PDG activator Pi (inorganic phosphate) has been increased. BCH stimulates glutamine utilization via GDH activation, thus providing the ATP signal necessary for insulin secretion. As denoted by the question marks, glutamate and/or glutamine seems to play some part in insulin degranulation by a yet-to-be-identified mechanism. EGCG blocks this process by inhibiting GDH activity. Furthermore, recent studies have also shown a link between mitochondrial GTP levels and insulin secretion, and GDH probably has some link to that as well. Abbreviations: AST, aspartate aminotransferase. (b) The location of some of the HI/HA mutants (green spheres) with respect to bound GTP.
occurs by binding to the active site, then it is not clear why Tetrahymena GDH is unaffected by leucine. Further, it is not clear how the unusual bicyclic heptane moiety of the activator BCH could fit into the relatively narrow pocket where that of the glutamate side chain normally binds. Therefore, it remains an open question as to where leucine is binding to exact its activation of the reaction. To directly ascertain which allosteric regulators require the antenna, the antenna was removed from human GDH and replaced with the short loop found in bacterial GDH. This should not directly affect GTP and ADP binding because none of the
ligand-contact residues reside on the antenna. However, the ‘antenna-less’ form of human GDH lost all forms of regulation except leucine activation. When the antenna from Tetrahymena GDH was spliced onto the main body of human GDH, this hybrid GDH exhibited all of the allosteric regulation found in human GDH. These results demonstrate that the antenna appears in the Ciliates and is necessary for allosteric regulation. One explanation for this evolutionary process is that allostery in GDH first evolved when the functions of the cellular organelles were changing (Figure 4). In the other 561
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Figure 3. Pharmaceutical lead compounds for treating HI/HA. (a) The effects of the four common polyphenols found in tea on GDH activity. (b) The structures of these compounds. (c) EGCG is efficacious against all HI/HA mutants of GDH and tetrahymena GDH (tGDH). However, it does not affect GDH activity if the antenna has been removed. (d) The in vitro activity of these compounds corresponds to the inhibition of BCH-stimulated insulin secretion in the perifusion assays.
eukaryotic organisms, all fatty-acid oxidation occurs in the peroxisomes [51,52]. In the Ciliates, fatty-acid oxidation is shared between the peroxisomes and the mitochondria [53,54] and, eventually, in animals, all of the mediumand long-chain fatty-acid oxidation moved into the mitochondria [55,56]. From the pattern of regulation in Ciliates, it seems that if the mitochondria have sufficient levels of fatty acids then the catabolism of amino acids is blocked. Only when the mitochondria are in a low-energy state (high ADP) will amino acids be catabolized. From the results of the studies on HI/HA, it seems that animals subsequently layered on additional regulation to link GDH activity with insulin homeostasis. Thus, GDH is activated when amino acids (protein) are ingested to promote insulin secretion and appropriate anabolic effects on peripheral tissues; in the glucose-fed state, GDH is inhibited in the pancreas perhaps to redirect amino acids into glutamine synthesis to amplify insulin release. Similarly, adjustment of hepatic GDH enables amino acid degradation to be suppressed when other fuels, such as fatty acids, are available but to be increased when protein (amino acids) are ingested and surplus amino acids can be oxidized. To this end, mammals developed leucine 562
activation and GTP inhibition while using the antenna architecture created by the Ciliates. The choice of leucine as a regulator is probably not an accident, because leucine is the most abundant amino acid in protein (10%) and provides a good measure of protein abundance. Similarly, the marked sensitivity of GDH for GTP over ATP is also not likely to be accidental. Most of the ATP in the mitochondria is produced from oxidative phosphorylation that is driven by the potential across the mitochondrial membrane created by NADH oxidation. Therefore, the number of ATP molecules generated from one turn of the tricarboxylic acid (TCA) cycle can vary between 1 and 29. By contrast, one GTP is generated per turn of the TCA cycle and, therefore, with the slow mitocondria–cytoplasm exchange rate, the GTP:GDP ratio is a much better measure of TCA-cycle activity than the ATP:ADP ratio. Indeed, recent results have demonstrated that mitochondrial GTP, but not ATP, regulates glucose-stimulated insulin secretion [57]. This is also consistent with the HI/HA disorder in that, without GTP inhibition of GDH, glutamate will be catabolized in an uncontrolled manner, the TCA cycle will generate more GTP and more insulin will be released. Therefore, the addition of GTP and leucine regulation to GDH makes it
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Figure 4. A model for the evolution of GDH allostery. This figure shows that the evolution of GDH allostery might be a form of exaptation [58]. Here, the antenna and some regulation could have been created in Ciliates in response to the evolution organelle function. These features might have then been further adapted for a different function, insulin homeostasis, in animals.
acutely sensitive to amino acid and glucose catabolism with obvious implications for insulin homeostasis. The results presented here have emphasized the evolution and role of GDH regulation in insulin homeostasis. However, some of the symptoms of the HI/HA syndrome also lend insight into the role of GDH in the brain and liver. Patients with HI/HA have two to five times higher levels of serum ammonium than normal patients [45]. The most likely explanation for this is that the hyperactive GDH catabolizes glutamate that not only releases ammonium but also depresses the production of N-acetylglutamate, a required allosteric activator of the first step in ureagenesis. Interestingly, these patients do not exhibit central nervous system (CNS) pathology normally associated with these serum levels of ammonium. In the case of the CNS, glutamate serves as an excitatory neurotransmittor. Glutamate is also the precursor of the inhibitory neurotransmitter g-aminobutyric acid (GABA), in addition to glutamine, a potential mediator of hyperammonemic neurotoxicity. Therefore, it might be that the disregulated GDH activity in the liver that causes the elevated ammonium levels could also protect the brain from the very same ammonium toxicity by catabolizing glutamate. The more subtle inference from all of these results is that GDH in animals probably favors the oxidative deamination direction but seems to be normally in a substantially inhibited state. The functional evolution of GDH was made possible by the dynamic nature of the enzyme. GDH necessarily undergoes the large domain movements to dehydrate the substrate and coenzyme to improve catalytic efficiency. The easiest way to regulate GDH, therefore, is to control these dynamic processes. In doing so, new functions and regulatory networks have been layered onto GDH through increasing the complexity of allosteric regulation rather than creating entirely new gene products. On the face of it, this tangled network of allosteric regulation is seemingly overly complex for an enzyme involved in such a mundane chemical reaction; it is, in fact, remarkable that this regu-
lation can adequately control a single enzyme that is apparently crucial to insure appropriate levels of glutamate in the brain, ureagenesis in the liver and insulin secretion in the pancreas. Acknowledgements This work was supported by National Institutes of Health (NIH; www.nih.gov) Grant DK072171 (to T.J.S.), NIH Grant DK53012 and American Diabetes Association (http://www.diabetes.org) Research Award 1–05-RA-128 (to C.A.S.) and NIH Grant DK19525 for islet biology and radioimmunoassay cores.
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Untangling the glutamate dehydrogenase allosteric nightmare Thomas J. Smith1 and Charles A. Stanley2 1 2
Donald Danforth Plant Science Center, 975 North Warson Road, Saint Louis, MO 63132, USA Endocrinology Division, Abramson Research Center, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the ‘missing link’ in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion. Fifty years of GDH studies Glutamate dehydrogenase (GDH) is found in all living organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NADP+ as the coenzyme [1]. This homohexameric enzyme has subunits comprising 450 and 500 amino acids in bacteria and animals, respectively. 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 Michaelis constant (Km) for ammonium in the reductive amination reaction seems to prohibit the reverse reaction under normal conditions in most organisms [2]. Even in plants, recent 15N-incorporation studies in the presence of excess ammonium have shown that GDH functions in the oxidative deamination reaction [3]. However, some bacteria use GDH rather than the normal glutamine synthetase–glutamate synthase pathway to fix nitrogen under high ammonia conditions [4]. The work reviewed here lends extremely strong support for GDH mainly operating in the oxidative deamination reaction in animals. Under most in vitro conditions, coenzyme release is the rate-limiting step, particularly at higher substrate concentrations [5]. From the properties of the most important allosteric regulators noted later, it seems highly likely that product release is the rate-limiting step in vivo as well. The crystal structures of the bacterial [6–8] and animal forms [9,10] of GDH have shown that the locations of the catalytically important residues have remained Corresponding author: Smith, T.J. ([email protected])
unchanged throughout epochs of evolution. However, GDH from animals, but not other kingdoms [11], is allosterically regulated by a wide array of ligands [11–17] (Table 1). GTP [17–19] (and, with 100-fold lower affinity, ATP [11]), is a potent inhibitor of the reaction and functions by increasing the binding affinity for the product, thereby slowing down enzymatic turnover [19]. Hydrophobic compounds such as palmitoyl coenzyme A (CoA) [20], steroid hormones [21] and steroid hormone analogs such as diethylstilbestrol (DES) [13] are also potent inhibitors. ADP is an activator of GDH [11,14,18,19,22], which functions 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 [16]. Its activation is akin to ADP but it acts at a site distinct from ADP [23]. Mammalian GDH exhibits several unusual kinetic properties with unclear physiological roles. Negative cooperativity is observed as nonlinear Lineweaver-Burk plots when NADP+ is varied [24]. Subsequent studies demonstrated that coenzyme (NADPH) binding to the initial subunits weakens the affinity to subsequent subunits [25,26]. This process is dependent upon the substituent at the a carbon of the substrate backbone [27]. The physiological role for this behavior has been suggested to maintain a particular catalytic rate or responsiveness as coenzyme concentrations vary in the mitochondria [28]. However, it is also possible that negative cooperativity is a consequence of inter-subunit communication necessary for other purposes. This communication has been shown by abrupt and striking changes in the circular dichroism and fluorescence spectra when GDH is half saturated [26] and from chemical modification studies demonstrating that the loss of enzymatic activity is disproportional to the number of subunits modified [29–31]. There is marked substrate inhibition in both reaction directions that is exacerbated by GTP and antagonized by ADP [14]. This might be more indicative of product release being the rate-limiting step rather than a form of regulation per se. GDH also has a second binding site for NADH that has been suggested to inhibit the enzyme and bind synergistically with GTP [12,32,33]. However, this inhibition only occurs at high, non-physiological concentrations. Animal GDH has been extensively studied since the early 1950 s. However, the mechanisms of allostery and the reason why animals created such a tangled network of regulation has been unclear until very recently. Structural studies have started to deconvolute mammalian GDH allostery, its possible evolutionary origins and, from
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Table 1. Summary of GDH regulation Type of regulation Regulator GTP Inhibition Palmitoyl CoA NADH (second site) ATP Diethylstilbestrol (DES) Zn2+ Leucine Activation BCH ADP Negative cooperativity Substrate inhibition
Coenzyme Glutamate 2-oxoglutarate
Behavior Increases coenzyme affinity and stabilizes abortive complex. Antagonist with ADP. Longer fatty acids are more effective than shorter chains. Inhibits at high concentrations. Agonist with GTP. Similar to, but 100 times weaker efficacy than, GTP. Inhibition reversed by ADP and leucine. Reversed by ADP and stabilizes abortive complex. Also a substrate. Activation differs to ADP but also an antagonist to DES and GTP. Non-hydrolysable form of leucine that also activates the enzyme. Decreases affinity for coenzyme and abortive complex. Antagonist for GTP. Can inhibit at low coenzyme concentrations. Observed in binding studies for all forms, observed kinetically for NADP+. Second position substituent on glutamate backbone is essential. Strongest at higher pH. Strongest at lower pH.
analysis of a severe hyperinsulinemic hypoglycemia disorder, demonstrate the importance of GDH allosteric regulation in humans. The structure of mammalian GDH The structure of mammalian GDH (Figure 1) is two trimers of subunits stacked directly on top of each other with each subunit being composed of at least three domains [9,10,34,35]. 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. Rising above these two domains is a long protrusion, ‘antenna’, that is a helix– loop–helix composed of 50 residues, which is not found in bacteria, plants, fungi and the vast majority of protists. The antenna from each subunit lies immediately behind the adjacent, counter-clockwise neighbor within the trimer. That these intertwined antennae are only found in the forms of GDH allosterically regulated by numerous ligands leads to the obvious conclusion that it plays a major part in this regulation. By mixing the coenzyme from one side of the reaction with a substrate from the other side of the reaction, it is possible to saturate GDH with ‘abortive’ or ‘dead-end’ complexes. When compared with the apo form of the enzyme, it is clear that GDH performs a complex ballet during catalytic turnover [9,10,34,35] (Figure 1). The substrate binds to the deep recesses of the cleft between the NAD-binding domain and the lower domain. The coenzyme binds along the NAD-binding-domain surface of the cleft. Upon binding, the NAD-binding domain rotates by 188 to firmly close down upon the substrate and coenzyme (Figure 1; arrow 1). As the catalytic cleft closes, the base of each of the long ascending helices in the antenna seems to rotate out in a counter-clockwise manner to push against the ‘pivot’ helix of the adjacent subunit (Figure 1; arrow 2). There is a short helix in the descending loop of the antenna that becomes distended and shorter as the mouth closes in a manner akin to an extending spring (Figure 1; arrow 3). The pivot helix rotates in a counter clockwise manner along the helical axes in addition to rotating counter clockwise around the trimer threefold axis. Finally, the entire hexamer seems to ‘exhale’ as the mouth closes (Figure 1; arrow 4). The three pairs of subunits that sit on top of each other move as rigid units towards each other, compressing the cavity at the core of the hexamer. 558
Refs [17–19] [20] [12,32,33] [11] [20] [61] [16] [15,62] [11,14,18,19,22] [24–26] [14] [14]
Structural mechanisms of allosteric regulation From the structures of various GDH–ligand complexes (Figure 1), GDH allosteric regulation is likely to be correlated with these complex motions. GTP binds to the side of the ‘jaw’ of each subunit (Figure 1c). As the NAD-binding domain closes, a space is opened between the pivot helix and the back of the domain. This affords enough space for GTP to bind mainly via interactions with its triphosphate moiety [9,34]. This explains why GTP increases the affinity for coenzyme; product release is made more difficult. By contrast, ADP binds behind the NAD-binding domain (Figure 1b) immediately under the pivot helix [35]. ADP cannot activate by locking the catalytic mouth open because that would also prevent catalytic turnover. Instead, it was proposed that ADP activates by helping the mouth to open by interacting via a basic residue on the pivot helix (Arg463) [35]. Indeed, if Arg463 is mutated to alanine, the enzyme is still able to bind ADP but is no longer activated by it [35]. This gives a physical explanation for the ADP and GTP antagonism in that they have opposite effects on the motion of the NAD-binding domain and also indirectly affect each other’s binding sites. At the physiological level, these two ligands are acting as energy sensors. When the mitochondria are at a high energy state and rich in triphosphates, GDH is inhibited. When the mitochondria are low in energy, the elevated ADP levels activate GDH to catabolize glutamate and feed the Krebs cycle with 2-oxoglutarate. The ADP and GTP antagonism indicates that GDH is kept in a tense state in which activity is finely tuned depending on subtle changes in the ADP:GTP ratios. The complex series of motion associated with catalytic turnover might also lend some clues as to how negative cooperativity is exacted. Because the ADP and apo forms of the enzyme are in the ‘open’ state, it seems safe to assume that this conformation represents the lowest energy. Therefore, perhaps increasing amounts of energy are required to saturate the enzyme as the enzyme is forced into the more compressed protein conformation. In the case of animal GDH, this unfavorable energy probably comes from a combination of the changes in the antenna, the position of the NAD-binding domain or the compression of the core. An exciting prospect is that perhaps some, or all, of these changes suddenly ‘snap’ into place as the enzyme becomes saturated, as indicated by the previous spectroscopy experiments [26]. A larger question is what is
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Figure 1. Structure and regulation of GDH. (a) The apo form of bovine GDH with each of the identical subunits shown in different colors. The light and dark hues show the dimeric pairing of the subunits, and these dimers move in and out as the catalytic mouth closes and opens. The arrows show regions of motion during catalytic turnover. As the NAD-binding domain closes on the substrate and coenzyme (arrow 1), the antenna from the adjacent subunit pushes against the pivot helix (arrow 2), a helix in the descending strand of the descending strand of the antenna becomes distorted (arrow 3) and the core of the enzyme compresses (arrow 4). (b) A close up of the ADP (green spheres) complex. Also shown is the side-chain of Arg463 (mauve) that, when mutated to an alanine, abrogates ADP activation. (c) Bovine GDH complexed with NADPH (gray), glutamate (orange) and GTP (mauve).
the advantage of negative cooperativity when the enzyme could have been made as a single subunit or with the subunits in more isolated environments? One possibility is that negative cooperativity might make the enzyme more efficient by transferring the energy associated with substrate binding to one subunit to facilitate product release from other subunits [36]. This is akin to a car engine where the explosion in one cylinder compresses the gas/air mixture in another. Because some of these motions have been observed in the more primitive forms of GDH [7,37], such conservation of binding energy could have been part of the original evolutionary design of this
enzyme. The following discussion focuses on the mechanisms and evolution of regulation by ligands binding to sites other than the active site (hence the Greek word allostery; allos ‘other’, stereos ‘shape’) that appeared along the way to the evolution of animal GDH (Box 1). The role of animal GDH allostery Although the structures have defined the locations of many of the allosteric sites, the reason for the sudden appearance of regulation in animals was not at all clear. A clue to this puzzle came from studies that linked GDH regulation with insulin and ammonia homeostasis. The mitochondrion of 559
Review Box 1. Models of allosteric regulation There are two widely accepted models for the induction of allostery in oligomeric proteins, the Monod-Wyman-Changeux (MWC) model [59] and the Koshland-Nemethy-Filmore (KNF) model [28,60]. The MWC model describes the positive cooperative binding of oxygen to hemoglobin by indicating that the subunits are in equilibrium between a low-affinity (T) and a high-affinity (R) state. Added ligand will bind to the R state and the equilibrium will adjust by moving more of the T-state subunits to the R state. In this way, the ligand binds in a positively cooperative manner; the affinity of the ligand for the protein increases with increasing ligand concentration. However, this model cannot explain situations in which ligands bind in a negatively cooperative manner. Alternatively, the KNF model indicates that ligand binding to one subunit can induce conformational changes in the other subunits resulting in either an increase (positive cooperativity) or decrease (negative cooperativity) in ligand affinity. The results reviewed here indicate that allosteric regulation of GDH probably occurs via a mechanism most in line with the KNF model. The homotrophic allostery observed with negatively cooperative binding of coenzyme clearly is well described by the KNF model. However, the heterotrophic allostery represented by GTP and ADP regulation is not exactly as described by KNF. It is clear that the antenna is involved and, therefore, it seems likely that there is communication among the subunits. However, the structural studies do not show that these allosteric regulators induce discreet conformation changes but, rather, it seems more likely that they are affecting the amount of energy required for the domain motion required for catalysis.
the pancreatic b-cell have an integrative role in the fuel stimulation of insulin secretion (Figure 2). The current concept is that mitochondrial oxidation of substrates increases the cellular phosphate potential that is manifested by a rise in the ATP4–:MgADP2– ratio. This, in turn, closes the plasma membrane KATP channels to depolarize the membrane potential, opens voltage-gated Ca2+ channels, causes a rise of free cytoplasmic Ca2+ and leads to insulin granule exocytosis. The connection between GDH and insulin regulation was initially established using a nonmetabolizable analog of leucine [15,38], BCH (b-2-aminobicyclo[2.2.1]-heptane2-carboxylic acid). These studies demonstrated that activation of GDH was tightly correlated with increased glutaminolysis and release of insulin. In addition, it has also been noted that factors that regulate GDH also affect insulin secretion [39]. It was suggested that glutamate serves as a mitochondrial intracellular messenger when glucose is being oxidized and that the GDH participates in this process by synthesizing glutamate [40]. However, as discussed before, the hypothesis that GDH (with a very high Km for ammonium) functions in the reductive amination reaction in vivo is controversial [41]. Subsequently, it was postulated that glutamine could also have a secondary messenger role and that GDH plays a part in its regulation [42–44]. The in vivo importance of GDH in glucose homeostasis was demonstrated by the discovery that a genetic hypoglycemic disorder, the hyperinsulinemia/hyperammonemia (HI/HA) syndrome, is caused by loss of GTP regulation of GDH [45–47]. Therefore, allosteric regulation of GDH is central to understanding the response of the b-cell to the cellular fuel state. Children with HI/HA have increased b-cell responsiveness to leucine and susceptibility to hypoglycemia after highprotein meals [48]. This is likely to be due to uncontrolled 560
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catabolism of amino acids yielding high ATP levels that stimulate insulin secretion and high serum ammonium levels. The elevation of serum ammonia levels reflects the consequence of altered regulation of GDH in liver, leading to increased ammonia production from glutamate oxidation and, possibly, also impaired urea synthesis by carbamoylphosphate synthetase due to reduced formation of its activator, N-acetyl-glutamate, from glutamate. This genetic lesion disrupts the regulator linkage between glycolysis and amino acid catabolism. During glucose-stimulated insulin secretion in normal individuals, it has been proposed that the generation of high-energy phosphates inhibits GDH and promotes conversion of glutamate to glutamine, which, alone or combined, might amplify the release of insulin [42,43]. Further support for this contention came from studies on the inhibitory effects of the polyphenolic compounds (Figure 3) from green tea on BCH-stimulated insulin secretion [49]. In these studies, normal b-cells are first ‘run down’ such that they are at a low-energy state (low GTP and ATP levels). When BCH is added, GDH is stimulated to catabolize glutamate, 2-oxoglutarate is formed and fed into the Krebs cycle, ATP levels are increased and insulin is released. Of the four prevalent polyphenols found in green tea [epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epicatechin (EC) and epigallocatechin (EGC)] only EGCG and ECG inhibited both wild-type and HI/HA mutant forms of GDH activity in vitro. The notable exception was that EGCG did not inhibit GDH if the antenna had been removed. This specificity carried over into the perfused-islet studies that demonstrated that EGCG, but not EGC, blocked GDH-mediated insulin secretion. This not only lends support for the contention that GDH has an important role in insulin homeostasis but also indicates that the HI/HA disorder might be directly controlled pharmaceutically. Evolution of GDH The structure of mammalian GDH shows that the 50residue antenna represents a clear departure from its ancestral forms and is part of the concerted movement of the enzyme subunits that occurs during catalytic turnover. However, it was not initially clear when and why this feature suddenly evolved. Recent genomics work has shown that the Ciliates within the Protista have an antenna that is slightly smaller than that found in the Animalia kingdom in spite of the fact that other members of the Protista, such as trypanosomes, have GDH nearly identical to bacterial forms [50]. Therefore, the Ciliates are an unexpected evolutionary ‘missing link’ in GDH development. To try to address the reasons for the evolution of the antenna, the allosteric behavior of several forms of GDH were examined [50]. With regard to regulation, Tetrahymena GDH is, indeed, between kingdoms; it is activated by ADP and inhibited by palmitoyl CoA, but unaffected by GTP and leucine. Interestingly, the lack of leucine activation of Tetrahymena GDH indicates, but does not prove, that there could, indeed, be a secondary binding site for leucine. The catalytic site is highly conserved between the GDHs from the various sources. Therefore, if activation
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Figure 2. Link between GDH and insulin homeostasis. (a) The role of GDH in BCH-stimulated insulin secretion and the effects of EGCG on this process. In energy-depleted bcells, a BCH ramp stimulates insulin secretion. Here, the most importnant energy source is glutaminolysis via phosphate-dependent glutaminase (PDG) and GDH, because the concentration of GDH inhibitors (ATP and GTP) has been depleted and the PDG activator Pi (inorganic phosphate) has been increased. BCH stimulates glutamine utilization via GDH activation, thus providing the ATP signal necessary for insulin secretion. As denoted by the question marks, glutamate and/or glutamine seems to play some part in insulin degranulation by a yet-to-be-identified mechanism. EGCG blocks this process by inhibiting GDH activity. Furthermore, recent studies have also shown a link between mitochondrial GTP levels and insulin secretion, and GDH probably has some link to that as well. Abbreviations: AST, aspartate aminotransferase. (b) The location of some of the HI/HA mutants (green spheres) with respect to bound GTP.
occurs by binding to the active site, then it is not clear why Tetrahymena GDH is unaffected by leucine. Further, it is not clear how the unusual bicyclic heptane moiety of the activator BCH could fit into the relatively narrow pocket where that of the glutamate side chain normally binds. Therefore, it remains an open question as to where leucine is binding to exact its activation of the reaction. To directly ascertain which allosteric regulators require the antenna, the antenna was removed from human GDH and replaced with the short loop found in bacterial GDH. This should not directly affect GTP and ADP binding because none of the
ligand-contact residues reside on the antenna. However, the ‘antenna-less’ form of human GDH lost all forms of regulation except leucine activation. When the antenna from Tetrahymena GDH was spliced onto the main body of human GDH, this hybrid GDH exhibited all of the allosteric regulation found in human GDH. These results demonstrate that the antenna appears in the Ciliates and is necessary for allosteric regulation. One explanation for this evolutionary process is that allostery in GDH first evolved when the functions of the cellular organelles were changing (Figure 4). In the other 561
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Figure 3. Pharmaceutical lead compounds for treating HI/HA. (a) The effects of the four common polyphenols found in tea on GDH activity. (b) The structures of these compounds. (c) EGCG is efficacious against all HI/HA mutants of GDH and tetrahymena GDH (tGDH). However, it does not affect GDH activity if the antenna has been removed. (d) The in vitro activity of these compounds corresponds to the inhibition of BCH-stimulated insulin secretion in the perifusion assays.
eukaryotic organisms, all fatty-acid oxidation occurs in the peroxisomes [51,52]. In the Ciliates, fatty-acid oxidation is shared between the peroxisomes and the mitochondria [53,54] and, eventually, in animals, all of the mediumand long-chain fatty-acid oxidation moved into the mitochondria [55,56]. From the pattern of regulation in Ciliates, it seems that if the mitochondria have sufficient levels of fatty acids then the catabolism of amino acids is blocked. Only when the mitochondria are in a low-energy state (high ADP) will amino acids be catabolized. From the results of the studies on HI/HA, it seems that animals subsequently layered on additional regulation to link GDH activity with insulin homeostasis. Thus, GDH is activated when amino acids (protein) are ingested to promote insulin secretion and appropriate anabolic effects on peripheral tissues; in the glucose-fed state, GDH is inhibited in the pancreas perhaps to redirect amino acids into glutamine synthesis to amplify insulin release. Similarly, adjustment of hepatic GDH enables amino acid degradation to be suppressed when other fuels, such as fatty acids, are available but to be increased when protein (amino acids) are ingested and surplus amino acids can be oxidized. To this end, mammals developed leucine 562
activation and GTP inhibition while using the antenna architecture created by the Ciliates. The choice of leucine as a regulator is probably not an accident, because leucine is the most abundant amino acid in protein (10%) and provides a good measure of protein abundance. Similarly, the marked sensitivity of GDH for GTP over ATP is also not likely to be accidental. Most of the ATP in the mitochondria is produced from oxidative phosphorylation that is driven by the potential across the mitochondrial membrane created by NADH oxidation. Therefore, the number of ATP molecules generated from one turn of the tricarboxylic acid (TCA) cycle can vary between 1 and 29. By contrast, one GTP is generated per turn of the TCA cycle and, therefore, with the slow mitocondria–cytoplasm exchange rate, the GTP:GDP ratio is a much better measure of TCA-cycle activity than the ATP:ADP ratio. Indeed, recent results have demonstrated that mitochondrial GTP, but not ATP, regulates glucose-stimulated insulin secretion [57]. This is also consistent with the HI/HA disorder in that, without GTP inhibition of GDH, glutamate will be catabolized in an uncontrolled manner, the TCA cycle will generate more GTP and more insulin will be released. Therefore, the addition of GTP and leucine regulation to GDH makes it
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Figure 4. A model for the evolution of GDH allostery. This figure shows that the evolution of GDH allostery might be a form of exaptation [58]. Here, the antenna and some regulation could have been created in Ciliates in response to the evolution organelle function. These features might have then been further adapted for a different function, insulin homeostasis, in animals.
acutely sensitive to amino acid and glucose catabolism with obvious implications for insulin homeostasis. The results presented here have emphasized the evolution and role of GDH regulation in insulin homeostasis. However, some of the symptoms of the HI/HA syndrome also lend insight into the role of GDH in the brain and liver. Patients with HI/HA have two to five times higher levels of serum ammonium than normal patients [45]. The most likely explanation for this is that the hyperactive GDH catabolizes glutamate that not only releases ammonium but also depresses the production of N-acetylglutamate, a required allosteric activator of the first step in ureagenesis. Interestingly, these patients do not exhibit central nervous system (CNS) pathology normally associated with these serum levels of ammonium. In the case of the CNS, glutamate serves as an excitatory neurotransmittor. Glutamate is also the precursor of the inhibitory neurotransmitter g-aminobutyric acid (GABA), in addition to glutamine, a potential mediator of hyperammonemic neurotoxicity. Therefore, it might be that the disregulated GDH activity in the liver that causes the elevated ammonium levels could also protect the brain from the very same ammonium toxicity by catabolizing glutamate. The more subtle inference from all of these results is that GDH in animals probably favors the oxidative deamination direction but seems to be normally in a substantially inhibited state. The functional evolution of GDH was made possible by the dynamic nature of the enzyme. GDH necessarily undergoes the large domain movements to dehydrate the substrate and coenzyme to improve catalytic efficiency. The easiest way to regulate GDH, therefore, is to control these dynamic processes. In doing so, new functions and regulatory networks have been layered onto GDH through increasing the complexity of allosteric regulation rather than creating entirely new gene products. On the face of it, this tangled network of allosteric regulation is seemingly overly complex for an enzyme involved in such a mundane chemical reaction; it is, in fact, remarkable that this regu-
lation can adequately control a single enzyme that is apparently crucial to insure appropriate levels of glutamate in the brain, ureagenesis in the liver and insulin secretion in the pancreas. Acknowledgements This work was supported by National Institutes of Health (NIH; www.nih.gov) Grant DK072171 (to T.J.S.), NIH Grant DK53012 and American Diabetes Association (http://www.diabetes.org) Research Award 1–05-RA-128 (to C.A.S.) and NIH Grant DK19525 for islet biology and radioimmunoassay cores.
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