The activation loop and substrate-binding cleft of glutaminase C are allosterically coupled

Abstract

The glutaminase C (GAC) isoform of mitochondrial glutaminase is overexpressed in many cancer cells and therefore represents a potential therapeutic target. Understanding the regulation of GAC activity has been guided by the development of spectroscopic approaches that measure glutaminase activity in real time. Previously, we engineered a GAC protein (GAC(F327W)) in which a tryptophan residue is substituted for phenylalanine in an activation loop to explore the role of this loop in enzyme activity. We showed that the fluorescence emission of Trp-327 is enhanced in response to activator binding, but quenched by inhibitors of the BPTES class that bind to the GAC tetramer and contact the activation loop, thereby constraining it in an inactive conformation. In the present work, we took advantage of a tryptophan substitution at position 471, proximal to the GAC catalytic site, to examine the conformational coupling between the activation loop and the substrate-binding cleft, separated by ∼16 Å. Comparison of glutamine binding in the presence or absence of the BPTES analog CB-839 revealed a reciprocal relationship between the constraints imposed on the activation loop position and the affinity of GAC for substrate. Binding of the inhibitor weakened the affinity of GAC for glutamine, whereas activating anions such as P_i increased this affinity. These results indicate that the conformations of the activation loop and the substrate-binding cleft in GAC are allosterically coupled and that this coupling determines substrate affinity and enzymatic activity and explains the activities of CB-839, which is currently in clinical trials.

Introduction

The importance of glutamine metabolism in cancer cell survival has been attracting an increasing amount of attention, resulting in a renewed focus on developing therapeutic approaches that target glutamine metabolism in transformed cells (1, 2). As described elsewhere, glutaminase (GLS)⁵ can be viewed as a gateway enzyme for glutamine metabolism, as it is responsible for a majority of the glutamine to glutamate deamination in cells (3). The pivotal role for GLS in glutamine metabolism, coupled with the recognition that many cancer cell types exhibit a pronounced dependence on glutamine, has provided the impetus for the development of a number of inhibitors targeting GLS to attenuate glutamine metabolism, some of which have undergone clinical testing (4, 5). The mechanisms by which GLS is activated and inhibited by small molecules continue to be of great interest and provide the rationale for our development of spectroscopic probes that can monitor the conformational states of GLS induced by the binding of allosteric activators and inhibitors.

Tryptophan fluorescence in intact, active proteins can be used as a sensitive readout to monitor conformational changes induced upon their interactions with small molecules. Past applications of this approach include monitoring the nucleotide-bound state of GTP-binding proteins and GTP hydrolysis, as well as revealing the kinetics of protein folding (6–9). Tryptophan fluorescence has also been used as a probe to monitor the conformational changes in enzymes that are induced by the binding of allosteric inhibitors and activators (10, 11). In some cases, it has served as a direct readout for substrate binding and in this way, can probe the mechanisms of activation and inhibition of enzymes by monitoring their effects on substrate binding (12, 13).

The enzymatic activity of GLS has been shown to correlate with tetramerization, which is believed to occur via the activation loop (14, 15). Here, by substituting a tryptophan for a tyrosine residue at the GAC active site, we set out to investigate how an inhibitor versus an activator communicates with the glutamine-binding site via the activation loop. As described in previous work (14, 15), the activation loop serves a critical role in GLS activation and inhibition as both allosteric inhibitors and activators are observed to bind in its immediate vicinity. Within the glutamine-binding cleft at the active site, ∼16 Å away from this loop, there are three residues that constitute a catalytic triad critical for substrate binding as well as catalysis. A comparison of the X-ray structures of active WT GAC with bound glutamate (product), and an inactive glutaminase domain bound with glutamine (substrate), show that tyrosine 471 is unique in its interaction with the amide nitrogen of the side chain of glutamine (15–18; Fig. 1). The amide to carboxylic acid conversion that distinguishes glutamine and glutamate therefore suggested GAC(Y471W) as a potential tryptophan sensor for monitoring glutamine binding to the enzyme.

Figure 1.

X-ray crystal structure of WT GAC bound to the substrate glutamine. As one of the essential side chains at the catalytic site, Tyr-471 (yellow) forms a hydrogen bond with the amide nitrogen of glutamine. The phenylalanine 327 side chain (red) located in the activation loop is shown to illustrate the considerable distance between the glutamine-binding cleft and the tetramer interface.

In this study, we describe the enzymatic and spectroscopic properties of GAC(Y471W) and demonstrate that it provides a direct readout for monitoring glutamine binding to GAC. This allows for the determination of glutamine binding affinity in the presence and absence of GAC activators and inhibitors. The apparent communication between the activation loop at the tetramer interface with the GAC active site suggests that the activation loop regulates substrate affinity and thus enzyme activity.

Results

Active site tryptophan substitution at GAC tyrosine 471 as a reporter for glutamine binding

Examination of the active site of GAC reveals residue Tyr-471, a residue that is accessible to solvent, forming a hydrogen bond with the amide nitrogen of glutamine (Fig. 1). This interaction, as well as Ser-291 and Tyr-419, has been previously shown to be essential for catalytic activity (18). Because the tryptophan side chain is comparable in bulk to tyrosine, we chose to mutate Tyr-471 to tryptophan. Initially, fluorescence emission of the GAC(Y471W) mutant was analyzed before and after the addition of 20 mm glutamine. We found that this saturating concentration of glutamine induced a maximal quenching of the tryptophan fluorescence of the GAC(Y471W) mutant, whereas addition of an equivalent concentration of glutamate did not change the fluorescence emission of GAC(Y471W) (Fig. 2A). The tryptophan emission changes observed with l-glutamine were stereospecific, as no such change was detected for d-glutamine, as well as substrate-specific, as the same concentration of l-asparagine induced no observed change in tryptophan emission (not shown).

Figure 2.

Glutamine quenches the intrinsic fluorescence of the GAC(Y471W) mutant. A, GAC(WT) and GAC(Y471W) at a final concentration of 300 nm were incubated with either glutamine or glutamic acid at a final concentration of 20 mm for 10 min at room temperature and their tryptophan emission spectra (excitation at 285 nm) were collected. GAC(WT), pink; GAC(Y471W)(−Gln/−Glu), red; GAC(Y471W)(+Gln/−Glu), green; and GAC(Y471W)(−Gln/+Glu), blue. B, real-time fluorescence emission (excitation: 340 nm, emission: 460 nm) was monitored for a reaction mixture containing NAD⁺ (0.2 mm), GDH (7 units/ml), with either GAC(WT) or GAC(Y471W) at a final concentration of 50 nm, and either in the absence or presence of P_i (50 mm). Glutaminase activity was initiated by the addition of glutamine to a final concentration of 20 mm at the indicated time (arrow). GAC(WT)(+P_i), blue; GAC(WT)(−P_i), red; GAC(Y471W)(+P_i), green; GAC(Y471W)(−P_i), pink. C, GAC(Y471W) at a final concentration of 300 nm was added to reaction buffer containing 100 mm P_i and the fluorescence emission (excitation: 285 nm; emission: 340 nm) was monitored in real-time. Subsequently, either glutamine or glutamate was added at to a final concentration of 20 mm at the indicated time (arrow). D, fluorescence emission (excitation: 285 nm, emission: 340 nm) of 300 nm GAC(Y471W) or GAC(D391K,Y471W) was monitored in real-time. At the indicated time (arrow), 100 mm P_i was added. Subsequently, 20 mm glutamine was added at the indicated time (arrow).

Maximal enzymatic activity of the GLS isoforms requires the presence of inorganic phosphate (P_i) at concentrations of 50–100 mm (19). The dose-response of enzyme activation as a function of glutaminase activity correlates with the formation of tetramers by GLS (19). Real-time NADH fluorescence emission assays used to assess GAC activity revealed that GAC(Y471W) possesses neither basal nor phosphate-stimulated enzymatic activity (Fig. 2B). This indicates that the observed fluorescence quenching upon addition of glutamine to GAC(Y471W) represents substrate binding in real time without any accompanying catalysis.

We next used the GAC(Y471W) mutant for real-time assays monitoring the kinetics of glutamine binding. GAC(Y471W) specifically reads out the binding of substrate, as 20 mm glutamine, in the presence of 100 mm P_i, quenched tryptophan fluorescence by 50%, whereas the same concentration of glutamate resulted in no change in 340 nm emission (Fig. 2C). The observation that GAC(Y471W) distinguishes between glutamine and glutamate binding, by virtue of the differences between the enzyme's substrate and its deaminated product, provides a method for examining in detail the allosterism underlying the formation of the active, tetrameric GLS species and the alterations at the substrate-binding site that precede catalysis.

Mutation of Asp-391 to a lysine residue on GAC (GAC(D391K)) results in an enzyme that is a constitutive dimer with no catalytic activity, irrespective of whether or not P_i was present. We introduced the Y471W mutation into GAC(D391K) yielding the GAC (D391K,Y471W) double mutant. We confirmed by size-exclusion chromatography that GAC (D391K,Y471W) was a constitutive dimer (data not shown). No binding of glutamine, as monitored using intrinsic tryptophan fluorescence, was detected using this constitutively dimeric form of glutaminase (Fig. 2D). This demonstrates that engagement of two GAC dimers at the helical interface to form a tetrameric species is a prerequisite for substrate binding and any observed enzymatic activity.

Thus, the GAC(Y471W) allows for the specific monitoring of the substrate-binding step, uncoupled from other events associated with substrate deamination and product formation. Upon examining the available crystallographic data, a comparison of the glutamine- and glutamate-bound glutaminase structures suggests the likely molecular contact responsible for the glutamine-specific changes in tryptophan fluorescence is the amide nitrogen lone pair of electrons interacting with the tryptophan aromatic π-moiety. The sensitivity of the Y471W substitution to readout glutamine but not glutamate binding is therefore attributable to the interactions of the amine group from the glutamine side chain with the aromatic indole side chain of the substituted tryptophan (16).

In the absence of the allosteric activator P_i, GAC(Y471W) fluorescence was titrated with increasing concentrations of glutamine, and the fluorescence quenching was observed to be dose-dependent. As the glutamine concentration was increased from 5 to 40 mm, the extent of quenching induced by the addition of glutamine increased (Fig. 3A). Each glutamine addition reached equilibrium within 10 min, with lower concentrations of the substrate displaying slower binding kinetics than higher concentrations (5 mm glutamine binding was complete within 10 min compared with 40 mm glutamine which required ∼4 min). By plotting the normalized fluorescence intensity at equilibrium versus glutamine concentration, we were able to determine the K_d value for substrate binding to GAC to be ∼5.2 mm (Fig. 3B), which is comparable with the value of the K_m (4.7 mm) determined from an initial rate analysis of activity, as shown in Fig. 3C.

Figure 3.

GAC(Y471W) is a spectroscopic reporter of bound substrate (glutamine) for the GAC enzyme. A, fluorescence emission (excitation: 285 nm, emission: 340 nm) of 300 nm GAC(Y471W) was monitored in real-time. At the indicated times (arrow), glutamine was added to the indicated final concentrations. B, equilibrium fluorescence from panel A is plotted as a function of glutamine concentration. A nonleast square fit of the data to Equation 1 (solid line) yielded an apparent K_d of 5.2 ± 1.1 mm (± S.E. of the fit). C, initial velocity of glutaminase activity in the absence of phosphate. Initial velocities of glutamine hydrolysis by GAC(WT) were measured at the indicated glutamine concentrations using the two-step assay. Each data point shown is the mean ± S.E. from 3 independent experiments. A simultaneous nonleast square fit of data from 3 independent experiments to Equation 2 (solid line) yielded a K_m of 4.7 ± 1.1 mm (± S.E. of the fit). D, fluorescence emission (excitation: 285 nm, emission: 340 nm) of 300 nm GAC(Y471W) was monitored in real-time. At the indicated times (arrow), glutamic acid was added to the indicated final concentrations. Subsequently, glutamine was added to a final concentration of 5 mm at the indicated time (arrow).

As shown in Fig. 2A, GAC(Y471W) does not change its 340-nm emission following the addition of glutamate. However, the binding of glutamate can be detected through its inhibition of glutamine binding. Because both the substrate and product occupy the same binding pocket, we tested whether the binding of glutamate can also affect the binding of glutamine. We first performed a competitive binding assay between glutamine and glutamate. Fig. 3D shows the fluorescence signal change induced by the addition of 5 mm glutamine to 300 nm GAC(Y471W), with increasing concentrations of glutamate added prior to the addition of glutamine. Consistent with the competitive inhibition by product binding, as previously described for glutaminase (20), the amount of quenching of GAC(Y471W) fluorescence induced by addition of glutamine decreased as the concentration of glutamate in solution was increased. The difference in substrate and product affinities estimated from these competition dose-responses is >30-fold, consistent with the expected lower GLS affinity for the product of deamination (glutamate), which suggests negative feedback of glutaminolysis would likely only occur under conditions of excess glutamate accumulation (20).

Phosphate regulates GAC substrate affinity and glutaminase activity by promoting tetramer formation

A possible clue regarding how phosphate increases glutamine binding is provided by previous size exclusion chromatography-multiangle light scattering results indicating the ability of GAC to form both dimeric and tetrameric species (21). The effect of phosphate observed in the size exclusion chromatography-multiangle light scattering experiments was to stabilize the tetrameric form of GAC, thus shifting the GAC distribution toward the higher oligomeric state (21). We then tested how the dimeric and tetrameric forms of GAC compare with regard to their ability to bind substrate. If dimeric GAC has no affinity for glutamine, then the observed higher affinity following phosphate addition may be due solely to the formation of the higher affinity, glutamine-binding tetrameric GAC species.

Fig. 4A demonstrates the effect of phosphate on the binding of glutamine to GAC(Y471W) in the WT background. Compared with the kinetics of glutamine binding in the absence of P_i, the kinetic traces shown in Fig. 4A demonstrate that under equivalent conditions (compare the 10 mm additions in Figs. 3A and 4A) the binding of glutamine in the absence of P_i is slower and results in less tryptophan quenching. At glutamine saturation, the quenching of GAC(Y471W) tryptophan fluorescence with 100 mm P_i present was ∼50%, comparable with the degree of quenching observed without P_i. This would suggest that the binding of glutamine, in the absence of phosphate, would stabilize tetramers and higher-order oligomers similar to those formed in the presence of P_i. As expected, GAC(Y471W) has been observed to form higher-order oligomers in the presence of glutamine when analyzed using size exclusion chromatography (data not shown). Although the nominal concentration of glutamine-binding sites is the same in each case (300 nm), in the absence of any binding by dimeric GAC, different values for the dissociation constant with or without P_i imply that the effective concentration of glutamine-binding sites is increased in the presence of P_i. This view is supported by the results of a titration of GAC(Y471W) tryptophan fluorescence carried out at a GAC concentration of 300 nm where a K_d of 1.1 mm, representing a 5-fold increase in the affinity of the enzyme for glutamine in the presence of P_i, was obtained (Fig. 4B). Significantly, under the same conditions, the K_m determined from an initial rate analysis with P_i is comparable to that measured in the absence of P_i (4.7 mm in both cases; Fig. 4C). Taken together, the differences in kinetic (i.e. K_m, V_max) and thermodynamic (K_d) parameters in the presence and absence of P_i can be accounted for by a phosphate-induced increase in glutamine-binding sites (see below).

Figure 4.

Effect of phosphate on the apparent binding affinity of glutamine for GAC(Y471W). A, fluorescence emission (excitation: 285 nm, emission: 340 nm) of 300 nm GAC(Y471W) was monitored in real-time in the presence of 100 mm P_i. At the indicated times (arrow), glutamine was added to the indicated final concentrations. B, equilibrium fluorescence from panel A is plotted as a function of glutamine concentrations. A nonleast square fit of the data to Equation 1 (solid line) yielded an apparent K_d of 1.1 ± 0.2 mm (± S.E. of the fit). C, initial velocity of glutaminase activity in the presence of phosphate. Initial velocities of glutamine hydrolysis by GAC(WT) was measured at the indicated glutamine concentrations in the presence of 100 mm P_i using the two-step assay method. Each data point shown is the mean ± S.E. from 3 independent experiments. A simultaneous nonleast square fit of the data from 3 independent experiments to Equation 2 (solid line) yielded a K_m of 4.7 ± 0.5 mm (± S.E. of the fit).

Effect of a small molecule inhibitor on glutamine binding to GAC(Y471W)

A number of allosteric inhibitors of GAC activity have been developed and shown to slow the growth of glutamine-dependent cancer cell lines (1, 2, 22). For one of these molecules, BPTES, there are several crystal structures that reveal the binding site for this and related inhibitors is at the activation loop at the GAC dimer-dimer interface. Until now, the precise molecular mechanism of inhibition was unclear. However, the GAC(Y471W) mutant made it possible to determine whether the mode of action of these inhibitors involves an inhibition of substrate binding.

To examine BPTES-like inhibitor effects on GAC(Y471W) tryptophan fluorescence, we used the allosteric inhibitor, CB-839, an analog of BPTES. CB-839 has been well-studied in vivo, recently undergoing clinical trials for treatment of triple negative breast cancer (5, 23). Similar to BPTES, CB-839 binds at the interface where two GAC dimers make contact to form a tetramer (16). To test the effect of CB-839 in the real-time fluorescence assay for glutamine binding, we added a substoichiometric amount of CB-839 (100 nm) to 200 nm GAC(Y471W), with the subsequent addition of 20 mm glutamine (Fig. 5A). Unlike multivalent anionic activators (i.e. phosphate, sulfate), which when added alone do not have any effect on the GAC(Y471W) fluorescence signal (e.g. see Fig. 2D), the addition of CB-839 alone resulted in a marked decrease in tryptophan fluorescence. Both the initial fluorescence quenching due to CB-839 and the subsequent quenching due to glutamine addition were dependent on CB-839 concentration (Fig. 5B) and inversely correlated (Fig. 5C). When normalized and plotted on the same scale, the data for the fractional quenching of GAC(Y471W) tryptophan emission by CB-839 (open triangles), and the fractional occupancy of glutamine-binding sites resulting from the subsequent addition of glutamine (closed circles), yielded similar IC₅₀ values for CB-839 of ∼30 nm. This range of values for the CB-839/GAC interaction is in good agreement with previous direct binding measurements (14) and K_i determinations from assays of enzyme activity (22). The results shown in Fig. 5C indicate that the inhibition of GAC by CB-839 is accomplished by decreasing the ability of GAC to bind glutamine.

Figure 5.

CB-839 inhibits GAC by reducing substrate affinity. A, fluorescence emission (excitation: 285 nm, emission: 340 nm) of 200 nm GAC(Y471W) was monitored in real-time. At the indicated times (arrow), DMSO (red) or 100 nm CB-839 (blue) were added. Subsequently, glutamine was added to a final concentration of 20 mm. B, fluorescence emission (excitation:285 nm) was monitored in real-time as in panel A. CB-839 was added to the indicated final concentrations. At the indicated times, glutamine was added to a final concentration of 20 mm. C, the equilibrium fluorescence from experiments performed as described in panel A after the addition of varying concentrations of CB-839 (open circles) or after the subsequent addition of glutamine (open triangles) was normalized and plotted as a function of CB-839 concentrations. Each data point shown is the mean ± S.E. average of 3 independent experiments. A nonleast square fit of the fluorescence quench due to CB-839 of Equation 3 yielded a K_0.5 of 37 ± 8 nm (± S.E. of the fit). A similar fit to the normalized glutamine binding yielded a K_0.5 of 23 ± 9 nm (±S.E. of the fit).

Discussion

Previous studies using the tryptophan fluorescence of the GAC(F327W) mutant, as a readout for conformational dynamics in the activation loop, demonstrated the utility of this approach for monitoring the binding of allosteric inhibitors and activators to the enzyme (21). Available structural data indicate that the activation loop of GAC and its active site are not in immediate proximity. This raises the question as to the mechanism by which activators and inhibitors that bind near the activation loop communicate with the active site. In addition to serving as a readout for inhibitor and activator binding at the activation loop, tryptophan fluorescence of the GAC(F327W) mutant also distinguishes the binding of substrate (glutamine) and product (glutamate) by displaying a greater fluorescence increase with the former. This observation is consistent with the view that there exists a tight reciprocal conformational coupling between the activation loop and the active site. Our previous results with GAC(F327W) indicated that glutamine binding at the active site affects the conformation of the activation loop and alters the microenvironment of GAC(F327W), which is reflected by the quenching of the fluorescent signal (see Fig. 6 where the two conformational states of Phe-327 are depicted). The nature of this intramolecular communication is of great interest as it promises to inform current and future efforts to develop next generation small molecule inhibitors that target glutaminase in glutamine-dependent cancer cells.

Figure 6.

A structural representation of the spatial relationships underlying changes in Y471W (yellow) tryptophan fluorescence that provide an assay to monitor the effects of CB-839 binding at the flexible activation loop in the vicinity of Phe-327. The two conformations of Phe-327 correspond to GAC bound to CB-839 (blue; dashed line represents the distance between the Phe-327 side chain and the amide nitrogen of bound glutamine of 16.1 A) or to phosphate (red; dashed line represents the distance between the Phe-327 side chain and the amide nitrogen of bound glutamine of 13.5 Å). Shown in ball and stick near Phe-327 is the inhibitor, CB-839.

The observation that the GAC(F327W) tryptophan fluorescence signal selectively responds to glutamine binding led us to ask whether a tryptophan substitution in the GAC active site could serve a similar role as a sensor to detect changes between substrate (glutamine) and product (glutamate) binding. Crystallographic data suggested that Tyr-471 was a good candidate because the side chain hydroxyl group of tyrosine forms a tetrahedral intermediate via a hydrogen bond with the amide group of the bound substrate glutamine, which is subsequently oxidized to the carboxyl group forming the product glutamate. The GAC(Y471W) mutant exhibits a significant amount of fluorescence quenching (about 50%) with the addition of 20 mm glutamine in the presence of phosphate, whereas no detectable quenching is observed when 20 mm glutamate is added to the same concentration of GAC(Y471W) (Fig. 2C). The observed difference in fluorescence quenching between substrate and product is likely due to the ability of the protonated amide nitrogen of glutamine to participate in a cation-π electrostatic interaction that results in tryptophan quenching, whereas the carboxyl group of glutamate cannot form bonds with the indole moiety of tryptophan 471 (26). Tryptophan does not have the hydroxyl group present in its side chain required to form the hydrogen bond with the substrate and position the H₂O molecule needed for the hydrolysis reaction. As a consequence, the Y471W mutant is able to bind glutamine but cannot carry out the catalytic deamination of glutamine to generate the glutamate product. The loss of catalytic activity is illustrated by the results from the real-time enzymatic assay of the Y471W mutant when compared with WT GAC, where the mutant did not show any catalytic activity even with the addition of 50 mm phosphate (Fig. 2B). The fact that the GAC(Y471W) mutant is catalytically defective is advantageous, as it allowed us to uncouple enzyme-substrate interactions from the actual catalytic event, thereby making it possible to monitor directly the substrate-binding step.

Taking this approach, we investigated whether different allosteric inhibitors and activators affect GAC activity by influencing glutamine binding. With phosphate present, the addition of the same concentration of glutamine to GAC exhibited more rapid binding and a greater degree of tryptophan quenching. Because phosphate acts to stabilize the active tetrameric species, we interpret the greater degree of quenching to reflect the increased number of available glutamine-binding sites in the presence of phosphate. This interpretation suggests a model where the conformational change that is induced by the formation of tetramers alters the GAC-substrate binding interaction. Based on earlier observations of phosphate-driven oligomerization of GAC (25), phosphate binding has the net effect of activating the enzyme by stabilizing its glutamine-binding, tetrameric state. Both tryptophan fluorescence and isothermal calorimetry experiments comparing WT GAC with the constitutively dimeric GAC(D391K), demonstrate that only the tetrameric form of GAC has the ability to bind glutamine (data not shown). Therefore, the observed phosphate-dependent enhancement of glutamine binding reflects the shift of GAC from dimer to tetramer upon the addition of phosphate. Under the conditions of the fluorescence assay (depicted in Fig. 4A), the concentration of GAC was typically 300 nm and therefore a mixture of dimer and tetramer. Addition of phosphate increases the amount of tetramer and shifts the size distribution of the GAC population toward the higher affinity, active enzyme complex. This result provides evidence to explain how P_i activates GAC by promoting high affinity substrate binding. The interactive nature of the activation loop and the enzyme active site suggests they are exquisitely coupled and reciprocally regulated in a reversible manner. This view is further supported by the observation that tetrameric and higher oligomeric species can be induced in WT GAC when size exclusion chromatography is performed in the presence of a saturating level of glutamine.⁶

For the BPTES-like inhibitors, GAC(Y471W) tryptophan fluorescence responded to the addition of the drug itself, which caused a dose-dependent quenching of fluorescence, that at drug saturation levels (∼100 nm) are ∼20% of the total fluorescence signal, allowing measurement of subsequent glutamine binding. The results are consistent with the observation made earlier that the conformational change of the activation loop can affect the active site and therefore would be predicted to influence the microenvironment of tryptophan 471. As shown in Fig. 5, B and C, when the concentration of the inhibitor CB-839 is increased, the tryptophan fluorescence change induced by the same amount of glutamine (20 mm) was significantly decreased. Taken together, these data suggest that CB-839, and other inhibitors of this class, attenuate GAC activity by allosterically inhibiting substrate binding. The physical details underlying the conformational communication between the activation loop and the enzyme active site remain unclear. The crystallographic data suggest that the mean distance between the proximal CB-839–binding site (i.e. the activation loop shown as Phe-327 in Fig. 6) and the active site is ∼16 Å, with no direct interaction between residues at each locus. Furthermore, on the distal side of the active site, there is a lid structure that allows access of glutamine (24). This further raises the question of how a constraining conformational change at the activation loop following CB-839 binding would provide a direct block of glutamine binding as has been proposed (15). However, there is good evidence for communication between the two sites via a peptide linkage, suggesting that a conformational change at the activation loop may result in changes in the glutamine-binding pocket that, in turn, regulate glutamine-binding affinity, giving rise to the observed effects on enzymatic activity (27).

In conclusion, the results presented here show that the GAC(Y471W) mutant provides a valuable tool for directly monitoring substrate binding to the enzyme. They demonstrate that the activation loop serves as a switch that regulates substrate affinity. Activators such as P_i position the switch to enhance glutamine binding, whereas inhibitors such as BPTES and CB-839 constrain the switch and block glutamine binding. This is summarized in the model depicted in Fig. 7, whereby activation of GAC entails a two-way conformational communication between the activation loop and active site substrate-binding cleft, with the ultimate outcome of determining glutamine affinity, which in turn, dictates GAC enzymatic activity.

Figure 7.

Schematic depicting how the oligomeric state of GAC, and the binding of activators (P_i) versus inhibitors (e.g. CB-839), influence the communication between the activation loop and catalytic site of GAC that determines substrate affinity and glutaminase activity. Note that both phosphate and BPTES class inhibitors stabilize tetramers but do so through different interactions with the activation loop. Not shown are tetramers formed by mass action alone in the absence of phosphate that possess activity although with a significantly lower V_max.

Experimental procedures

Recombinant protein expression

The recombinant GAC construct (Y471W) was expressed following a similar protocol that has been previously described (14). The mouse kidney-type glutaminase isoform 2 (GAC, NP_001106854.1) encoded plasmid (residues 72–603) was cloned into a pET28a vector with a N-terminal histidine tag and thrombin-cleavage site. The expressed protein was purified using Co²⁺ affinity beads, then followed by anion exchange and gel filtration chromatography. Purified GAC was stored at −80 °C in gel filtration buffer (20 mm Tris-HCl, pH 8.5, 500 mm NaCl, 1 mm NaN₃) after snap freezing in liquid N₂ for later use.

Fluorescence measurements

Fluorescence measurements were carried out on a Varian Cary Eclipse fluorimeter in counting mode. The sample was held in a 1-ml cuvette with continuous stirring at 20 °C. Stock solutions of glutamine (200 mm) and K₂HPO₄ (1 m) were prepared in reaction buffer (50 mm Tris acetate, pH 8.5, 0.1 mm EDTA). Tryptophan emission spectra were obtained by setting the excitation wavelength at 285 nm and scanning emission wavelengths from 310 to 460 nm. For kinetic experiments, the excitation and emission wavelengths were set at 285 and 340 nm, respectively. The chosen wavelengths provided the largest changes in tryptophan fluorescence when glutamine was added to GAC(Y471W). One hundred μl of a dilution of GAC(Y471W) were added to 0.9 ml of reaction buffer, either in the presence or absence of KH₂PO₄ (0.1 m) and the fluorescence emission was monitored in real time. Subsequently, 200 μl of diluted aliquots were added to achieve the indicated final concentrations of glutamine or glutamic acid. The kinetic assays in the presence of the inhibitor, CB-839, were also carried out using a similar procedure. Stock solutions of CB-839 were prepared in dimethyl sulfoxide (DMSO) as solvent and all dilutions were also prepared in DMSO. One hundred μl of a dilution of GAC(Y471W) were added to 0.9 ml of reaction buffer and fluorescence was monitored in real time. Subsequently, 20 μl of either DMSO or a dilution of CB-839 were added and the decrease in fluorescence was monitored. Once the fluorescence emission reached a constant value, 100 μl of 200 mm glutamine were added.

Real-time glutaminase assays

The real-time activity assays used to evaluate the activity of GAC mutants through the production of NADH were carried out using a Varian Cary Eclipse fluorimeter with the excitation and emission wavelengths set at 340 and 460 nm, respectively. Experiments were carried out in a 1-ml cuvette with continuous stirring at 20 °C. A 0.9-ml reaction buffer containing the appropriate concentrations of P_i was mixed with 10 μl of glutamate dehydrogenase (GDH) (700 units/ml) and 40 μl of NAD⁺ (50 mm). One hundred μl of an appropriate dilution of GAC were added to the reaction mixture and the fluorescence emission was monitored in real time. After 60 s, 100 μl of a glutamine solution were added (200 mm).

Two-step glutaminase assays

Glutaminase activity was measured using a two-step assay. In the first step, 77 μl of a reaction mixture containing either 100 mm or no P_i were prepared. Twenty μl of various dilutions of glutamine were added to the reaction mixture. Glutamine hydrolysis was initiated by the addition of 3 μl of GAC(WT) (10 μm stock). The reaction was allowed to proceed for 2 min at room temperature, after which it was quenched by the addition of 10 μl of 3 m hydrochloric acid and the samples were stored on ice. For the second step, a reaction mixture containing 130 mm Tris-HCl (pH 9.4), 7 units/ml of GDH, and 2 mm NAD⁺ was prepared. A 10-μl aliquot of each quenched sample from the first step was added to 200 μl of the reaction mix in a single well of a 96-well plate (Corning), mixed, and incubated at room temperature for 40 min. The absorbance at 340 nm was measured on a plate reader. The second reaction was carried out with varying concentrations of glutamic acid to obtain a standard curve. The slope of the linear part of the standard curve was used to convert the absorbance values to concentration of glutamine hydrolyzed. Initial velocities of glutamine hydrolysis were calculated by dividing the concentration of glutamine hydrolyzed by the time of the first reaction (2 min).

Data analysis

Dose-response curves of fluorescence as a function of glutamine concentration were fit to the following equation,

$$F=F_0-\left(F_0-F_f\right)\left[\frac{\sqrt{{\left(K_D+L_T+R_T\right)}^2-4R_T{L}_T}}{2R_T{L}_T}\right]$$ (Eq. 1)

where F is the equilibrium fluorescence attained upon the addition of glutamine at a concentration L_T, F₀ is the initial fluorescence before the addition of glutamine, F_f is the fluorescence attained in the presence of saturating concentrations of glutamine, R_T is the concentration of glutaminase used, and K_D is the dissociation constant.

Initial velocities of glutamine hydrolysis as a function of glutamine concentration were fit to the Michaelis-Menten equation,

$$V=\frac{V_{m}S}{K_m+S}$$ (Eq. 2)

where V is the initial velocity at any glutamine concentration, S is the concentration of glutamine, V_m is the maximal velocity at saturating concentrations of glutamine, and K_m is the Michaelis constant.

Dose-response curves of fluorescence as a function of CB-839 concentrations were fit to the following equation.

$$F=F_0+\frac{F_f-F_0}{1+\left(\frac{K_{0.5}}{I}\right)}$$ (Eq. 3)

where F is the equilibrium fluorescence attained upon the addition of CB-839 at a concentration I, F₀ is the initial fluorescence before the addition of CB-839, F_f is the fluorescence attained in the presence of saturating concentrations of CB-839, and K_0.5 is the concentration of CB-839 at which 50% of the fluorescence change occurs.

Author contributions

Y. L. and S. R. data curation; Y. L. and S. R. formal analysis; Y. L., S. R., T.-T. T. N., and C. A. S. investigation; S. R., C. A. S., and J. W. E. conceptualization; S. R., C. A. S., and J. W. E. methodology; S. R. and R. A. C. writing-review and editing; T.-T. T. N. validation; R. A. C. and J. W. E. supervision; R. A. C. funding acquisition; R. A. C. project administration; J. W. E. writing-original draft.