Substrate Activation and Conformational Dynamics of GMP Synthetase
Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. GMP synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of XMP. In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid ammonia transfer to this intermediate. Isolation and use of the adenylylated nucleotide intermediate enabled study of the amidotransfer reaction distinct from the ATP dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear faster than steady-state turnover, implying that release is likely rate limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites.
INTRODUCTION<!>Materials<!>Protein purification and steady-state assays<!>Stopped-flow Measurements<!>HPLC Assays<!>GMP synthetase Tryptophan to Phenylalanine Mutants<!>Analysis of Adenylylated XMP Intermediate<!>Nucleotide-only Stopped-flow Fluorescence Studies<!>Stopped-flow Fluorescence of Tryptophan to Phenylalanine Mutants<!>Nucleotide-only Stopped-flow Absorbance Studies<!>Stopped-flow Absorbance Studies with Glutamine<!>Steady-state turnover number<!>DISCUSSION
<p>Glutamine amidotransferases (GAT's) catalyze the amination of a wide range of molecules using the amide nitrogen of the side chain of glutamine; these enzymes consist of two domains, one that hydrolyzes glutamine, another that transfers the glutamine-derived ammonia to the waiting acceptor molecule (1). GMP synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the synthesis of GMP via the amination of XMP (Figure 1A) (1, 2). The two domains of the E. coli enzyme are contained on a single polypeptide, with a Class I glutamine amidotransferase domain contained in the first 208 residues and an N-type ATP-PPase domain in the remaining C-terminal segment.</p><p>The roles of the GMPS protein domain interactions in modulation of both catalytic and regulatory functions remain central to understanding the enzyme's broad biological functions. The central role of GMPS in primary metabolism offers examples of selective targeting of antimicrobial therapies (3). Virulence attenuation in M. tuberculosis has also generated focus on the mycobacterial GMPS (4). Furthermore, an antimetabolite therapy-resistant tumor phenotype is linked to both down-regulation of a single nucleotide point mutations in the GMPS guaA gene linked with reduced activation of 6-mercaptopurine and 6-thioguanine (5, 6). The significance of GMPS continues to emerge exemplified by the recent discovery of an allosteric role in gene regulation through histone deubiquitylation (7-9).</p><p>A common feature of amidotransferase catalysis is an activation step that prepares the acceptor molecule for amination (10). GMPS belongs to the class of N-type pyrophosphatases, which activate their various substrates by adenylylation (11). The substrate nucleotide, XMP, is adenylylated on the xanthine C2 oxygen (12), which is then primed for attack by a nitrogen nucleophile (Figure 1B). A previous study of GMPS demonstrated with positional isotope exchange that adenylylation is a reversible process, and, with steady state techniques, that substrate binding appears ordered, with ATP binding first, XMP second, and glutamine last (13).</p><p>Other common properties of amidotransferases are the conditional glutaminase activities. The glutamine hydrolysis function is not active until a suitable substrate acceptor occupies the distal second domain (1). Substrate binding leads to a series of conformational changes that propagate through the enzyme and culminate in glutaminase activation; the resulting ammonia is transferred via an intramolecular path to the waiting substrate (14). While the ammonia tunnel in some related enzyme systems has been elucidated (15, 16), the path between active sites in GMPS has remained undetermined despite the available crystal structures from several forms of the protein, including E. coli (17), H. sapiens (18), and Pyrococcus horikoshii (19). Limitations in these available structural data for GMPS warrant additional biophysical approaches for investigations aimed at addressing functional properties (17).</p><p>Recent studies with related members of the amidotransferase family indicate that upon binding of acceptor substrates, these enzymes undergo significant conformational changes (20-22). These conformational changes are associated with the formation of chambers or tunnels through which the released ammonia can travel between active sites (23-25). The allosteric steps that modulate glutaminase function continue to be the subject of study across a diverse set of amidotransferases (26-30). Distinctions within the different amidotransferase families exist, but the mechanisms for maintaining the fidelity of distal coupled activities are emerging.</p><p>GMPS has been reported to undergo a conformational change upon binding of its nucleotide substrates alone, without glutamine or other ammonia sources, as indicated by differential susceptibility to proteolysis or to chemical modification of surface cysteines (31, 32). Using distinct approaches, similar observations were recently made for the P. falciparum form of the enzyme (33); additional insights regarding glutaminase activation and ammonia utilization have also been recently gained for this enzyme (34). These studies provided evidence that GMPS can utilize exogenous ammonia in the presence of glutamine, while the exchange of glutamine-derived ammonia with bulk solvent was not observed, indicating the fidelity of the coupled catalytic processes. Even when exogenous ammonia is used as a substrate, there is a dependence on glutaminase and nucleotide acceptor site coupling relayed through conformational status of the protein.</p><p>The details of how the conformational states of GMPS relate to overall turnover events have not yet been established, although nucleotide binding is known to play a role. All current protein crystal structure data implicate a significant global conformational change in the monomeric subunit occurring during ammonia channel formation. The current study focuses on single-turnover events associated with the GMPS catalytic cycle to provide insights into the substrate activation and tunnel formation events that occur upon nucleotide binding. Using rapid reaction methodologies for single turnover experiments the direct observation has been made for the reaction intermediate of the GMPS reaction. In addition, a conformational change reported through a unique tryptophan residue has been observed during the catalytic cycle. These events have been now temporally aligned with the kinetics of a conformational change that occurs during turnover. The results provide the first complete kinetic cycle through direct observation of intermediate formation, glutamine hydrolysis and ammonia transfer in an amidotransferase.</p><!><p>Analytical-grade ATP, glutamine, and EPPS were obtained from commercial sources and used without further purification; commercial XMP was further purified as described (35). Nucleotide concentrations were determined by spectrophotometric measurement of solutions, with reference to published extinction coefficients (36). Water was processed through a commercial laboratory water purification system before use.</p><!><p>E. coli GMPS was overexpressed in E. coli DH5α and purified as described (37). Enzyme concentration was determined by dye-binding assay (38), using bovine serum albumin as standard. Specific activity of purified protein was determined by the direct assay, which continuously follows the 1500 M−1cm−1 decrease in extinction coefficient at 290 nm upon conversion of XMP to GMP (39). Enzyme preparations were homogeneous as determined by SDS-PAGE and by specific activity determinations (typically 40 μmole GMP/min/mg protein; data not shown). The turnover number (kcat) of the enzyme was determined by obtaining a substrate saturation curve at 25°C and fitting the data to the Michaelis-Menten equation with non-linear curve fitting. For these assays, glutamine substrate was varied; all others were held at saturating levels. Activity measurements for turnover number determination made use of the direct assay or of the endpoint glutaminase assay, as described (39, 40).</p><!><p>Stopped-flow measurements were obtained with a Hi-Tech SF-61DX2 instrument, using the single-mixing mode. The temperature was held at 25°C with a circulating water bath. Absorbance measurements were made using a 35W deuterium source and 1 cm path length configuration. In fluorescence measurements, a 75 W xenon-mercury arc lamp was used; the bandwidth of the excitation light was 2 nm full-width at half-height; emission light was passed through a 320 nm highpass filter. Instrument control and curve fitting were performed with the Hi-Tech KinetAsyst software, version 3.</p><p>For glutamine-free stopped-flow absorbance measurements, a solution of GMPS with ATP (typically 20 μM and 2 mM, respectively), was rapidly mixed with various concentrations of XMP. In measurements containing glutamine, XMP (120 μM) was pre-mixed with the enzyme and ATP at room temperature for 1-3 minutes before rapid mixing with a solution of glutamine (2 mM) and ATP. The concentrations given are those before one-to-one dilution in the stopped-flow device. In all cases, both solutions contained 25 mM EPPS (pH 8.5) and 10 mM MgCl2.</p><p>For fluorescence stopped-flow measurements, the enzyme concentration was kept to a final concentration of 0.25 μM to reduce inner-filter effects (35, 41) and to achieve pseudo-first order conditions. The concentrations of other components were maintained at levels given above. All stopped-flow measurements were the average of at least three and up to twenty replicate data collection events per titration point, except the 10 μM point in Figure 6C, which used two replicates. Each event collected 4096 points over the time course of the mixing.</p><!><p>In the analysis of enzyme-bound nucleotide intermediates, nucleotide-enzyme reactions (typically with 50-100 μM GMPS, 0.5 mM ATP, 200 μM XMP, 20 mM EPPS (pH 8.5), and 10 mM MgCl2) were mixed in a final volume of 100 μL and allowed to equilibrate at room temperature for 1 minute before addition of internal standard (100 μM CMP final concentration) followed by quenching and protein precipitation with ice-cold 50% trichloroacetic acid (2.5% of final volume). After incubation on ice for 5 minutes, protein was removed by centrifugation at 15,000 × g for 10 minutes at 4°C, the supernatant neutralized with 1 μL 10 N NaOH, and a 25 μL aliquot loaded onto a 4.6 × 250 mm (Alltech) or 4.6 × 150 mm (Supelco) C18 reversed-phase nucleotide separation HPLC column. Separation was achieved using 1 mL/min flow rate and a linear gradient from 5 to 22% acetonitrile over 10 min, then holding 22% for 15 minutes; both solvents also contained 50 mM KH2PO4 (pH 6.2) and 4 mM tetrabutylammonium phosphate. Detection was accomplished with a diode array detector, monitoring 220-320 nm. Fractions, collected manually, were heated in a sand bath heated to 100°C. Separation of authentic nucleotides revealed the following elution times with this solvent system: for the 250 mm column, CMP, 5.5 min; GMP, 6.9 min; AMP, 9.8 min; XMP, 13.3 min; ADP, 15.5 min; ATP, 17.2 min; for the 150 mm column, CMP, 3.4 min; GMP, 4.3 min; AMP, 6.1 min; XMP, 8.6 min; ADP, 10.7 min; ATP, 12.4 min.</p><!><p>Tryptophans to phenylalanine mutants were made using the QIAGEN Quickchange site-directed mutagenesis method. Two "mutagenizing" primers (i.e., containing the mutation of interest), complimentary to each other, amplified the entire plasmid containing the gene of interest, with the appropriate mutation. A silent mutation that added or removed a restriction site was included in the primers to simplify confirmation of correct plasmid construction. For tryptophan to phenylalanine (W to F) mutagenesis of the GMPS gene, the pET vector harboring the His-tagged E. coli GMPS was used as the template (Supplementary Materials).</p><p>The E.coli guaA contains eight tryptophans, which were removed one at a time using the primers shown in Table S1. The GMPS gene in this plasmid was flanked upstream by an NdeI site, downstream by a HinDIII site; in the middle of the gene is a SalI site. Two intermediate plasmids were made by sequential mutagenesis, one containing the first four W to F mutations, another containing the last four. The NdeI-SalI fragment containing the first four mutations and the SalI-HinDIII fragment containing the final four were combined onto one plasmid. Recovery of tryptophan at a particular position was achieved by the use of wild type primers for that position. The mutant proteins were purified as described above for the 6-histidine-tagged wild type protein (Supplementary Materials). During final buffer exchange of the protein, tryptophan mutants were observed to precipitate under low-salt conditions, but addition of NaCl to 100 mM eliminated this problem.</p><!><p>An earlier study suggested that the enzyme-bound adenylylated XMP intermediate is stable at least long enough to separate and analyze (12); however, no spectral information, which facilitates the interpretation of the pre-steady state results below, was reported for the compound. The availability of large amounts of enzyme from a recombinant system (37) facilitated the scale-up of the preparation of the intermediate for analysis. As expected, mixing of GMP synthetase with its nucleotide substrates, in the absence of an ammonia source, led to the formation of a new peak in the HPLC separation of nucleotides from the reaction (Figure 2A, lower trace); this peak was absent from the enzyme-free controls (data not shown). When the reaction mixture represented in the lower trace of Figure 2A was treated with glutamine, the new peak disappeared and the reaction product, GMP, appeared (Figure 2A, upper trace), consistent with the adenylylated XMP intermediate giving rise to the new peak in the lower trace. Furthermore, when this new peak was collected and boiled to dryness, it decayed into AMP and XMP, (Figure 2B). As the extinction coefficient of AMP is 1.9 times that of XMP at 260 nm (36), the peak areas of the breakdown products in Figure 2B indicate their presence in a 1:1 molar ratio. The UV-vis spectrum of the adenylylated XMP intermediate is dominated by the adenosine base, with some contribution in the lower ultraviolet from the xanthosine ring (Figure 2C). Attempts to quantify the intermediate by comparing it to decrease in substrate were inconsistent, so an estimate of the spectrum of adenylylated XMP has been converted in Figure 2C to millimolar absorptivity using the assumption that the intermediate would have approximately the same maximum as that of adenosine, which is 15.3 mM−1cm−1 at 259 nm (36). At 290 nm, the millimolar absorptivity of XMP is 4.8 mM−1cm−1, while adenylylated XMP is seen to be lower, at approximately 1.2 mM−1cm−1 (Figure 2C).</p><!><p>Several lines of study have concluded that GMP synthetase undergoes a significant conformational change upon binding of both of its nucleotide substrates, in the absence of an ammonia source (39). Indeed, stopped-flow mixing of GMP synthetase (premixed with ATP) with XMP substrate (in the absence of an ammonia source) gives rise to decay in the intrinsic tryptophan fluorescence of the enzyme (Figure 3A). This decay is attributed to a substrate-induced conformational change that alters the environment of one or more of the molecule's eight tryptophans, which are dispersed widely in the structure of the enzyme (17, 33). Although a lag phase is evident in these transients at early time points of less than 10ms, too few data points were available to obtain adequate curve fits to this phase. The apparent length of the lag phase did decrease with increasing nucleotide substrate concentration, suggesting the delay could be due to nucleotide binding event(s). Time points after the lag phase were modeled by a single-exponential curve of the type shown in Equation 1, where A is the amplitude of the change in fluorescence, R is the apparent rate constant, t is time, and C is the calculated final value of the fluorescence. The hyperbolic behavior of the fluorescence decay rate with increasing XMP concentration (Figure 3B) indicates that the step reported by the signal is a slower unimolecular isomerization or conformational change process occurring after a faster bimolecular binding event (42). A general mechanism consistent with such a two-step process is given in Scheme 1. In terms of the rate constants given in this Scheme, the equation describing such hyperbolic rate dependence is given in Equation 2 (42), where Ka = k−1/k+1. Fitting the data in Figure 3B to this equation provides estimates for Ka, k+2, and k−2; a tangent line to the curve at low substrate concentration provides an estimate for k+1, the apparent second-order rate constant for substrate binding, and thence also an estimate for k−1. The interpretation of the model above in the context of GMPS nucleotide binding events will be given below, as will the values of the estimates from this model.</p><!><p>E. coli GMPS has a total of eight tryptophan residues, greatly complicating the correlation of tryptophan fluorescence with specific conformational events in the protein. With the goal of simplifying the signal from these residues, several tryptophan to phenylalanine mutants were made. Although not all mutants have so far been generated, preliminary results were obtained that shed some light on the substrate-induced conformational response of the protein. The mutants discussed here relate to W37 in the protein, which, based on the structure of the enzyme, was thought to offer a position that could be in a "conformational hotspot," where the substrate-induced protein motions would alter the local environment of the residue. A "W37F" mutant was made, in which the W37 position was changed to a phenylalanine; a "37W" mutant was also made, in which all positions except the tryptophan at position 37 were changed to F; and lastly, a tryptophan deficient protein was generated, with all the W residues changed to F. Specific activities of the purified mutant proteins are shown in Table 2. Although the mutant proteins are less active than wild type, they still show significant capacity to turn over substrates, indicative of the overall retention of protein structure and function.</p><p>These proteins were examined with stopped-flow fluorescence, using similar conditions as described earlier with the wild type protein (Figure 4). For these data, all transients were collected on the same day, consecutively, to minimize differences between the datasets. Clearly the amplitude and the rate of the fluorescence decay vary with the mutation involved (Table 3). Although the tryptophan deficient mutant showed a slight change in amplitude upon mixing, this decay is not reported in this table. This change is likely the result of fluorescence changes from the remaining tyrosine residues in the protein and represents the background to the predominant tryptophan signal. Tryptophan-37 appears to be the primary reporter of conformational change in the protein, as it alone can lead to the majority of the fluorescence decay seen upon mixing with substrates.</p><!><p>Absorbance changes at 290 nm brought about by enzyme-catalyzed nucleotide transformations have long been used to monitor the GMPS reaction in the steady state(39). However, no pre-steady state observations of these nucleotide absorbance changes have been reported. Rapid mixing of GMPS-ATP with XMP gives rise to a visible decrease in 290 nm absorbance (Figure 5A). The rate and amplitude of the single-exponential curve fit to this absorbance change were clearly nucleotide concentration-dependent, demonstrating saturation at higher concentrations of XMP (Figure 5B). The decline in absorbance reaches a maximum rate of approximately 40 s−1, while at fixed protein concentration (10 μM), the magnitude of the 290 nm absorbance change reaches a maximum of approximately 3.5 × 10−2. Concentrations of XMP above 200 μM were not assayed, since GMPS shows substrate inhibition above this level (35). Similar to the fluorescence data, the behavior of the rate of the absorbance decrease is hyperbolic with increasing substrate, again implying that the observed change is due to a slow unimolecular step following a fast binding event. Unlike those from the fluorescence experiments, the data in this case were not fitted to the hyperbolic function, which requires pseudo-first order conditions; in most of the absorbance experiments, the reactions do not fulfill this requirement, since XMP substrate concentrations in these experiments ranged from nearly molar equivalence with GMPS, or up to only 20-fold excess. However, the rate of the absorbance decrease does appear to saturate at a maximum of approximately 40 s−1 (Figure 5B and 5C), independent of the protein concentration, while the magnitude of the decrease in absorbance is proportional to this variable (Figure 5C). A linear regression fit to the absorbance-change magnitude vs. GMPS concentration data gives a decline in absorbance of 3.1 × 10−3 per μM enzyme. The magnitude of change is consistent with the observed decline in 290 nm extinction coefficient upon adenylylation of XMP (Figure 2C), and is greater than the magnitude of the conversion of XMP to GMP (39). The respective values in nucleotide extinction coefficients are consistent with these results (Figure S1). These observations are consistent with the decrease in absorbance observed upon rapid mixing enzyme with XMP resulting from the chemical conversion of XMP to adenylyl-XMP. Given the observed absorbance changes and estimate of extinction coefficient for the adenylyl-XMP, 87% occupancy of the enzyme is observed.</p><p>Scheme 2 provides a mechanism that describes the kinetic steps occurring as GMPS encounters its nucleotide substrates, in the absence of an ammonia source. In accord with an earlier study (13), substrate addition is shown as ATP first, followed by XMP; all steps leading up to the adenylylated XMP are shown as reversible. The initial step of ATP-binding is included for completeness but was not investigated in this work. The two steps defined by k±1 and k±2, represent the binding of XMP substrate to the enzyme-MgATP complex followed by isomerization and conformational change. Since these steps are consistent with the general model given earlier for the fluorescence stopped-flow data, k±1 and k±2 are shared by Schemes 1 and 2. The values calculated for the estimated maximum rate of substrate-induced conformational change, as seen in the fluorescence stopped-flow experiments, is 104 s−1 (Table 1), approximately three-fold greater than the maximal rate of the chemical change, as conveyed by 290 nm absorbance. The chemical step of adenylylation is therefore reported as a discrete step, k3 (Scheme 2). In addition, an estimate for the rate of the k−3 reaction (return of adenylylated XMP to ATP and XMP), is implicit in an earlier positional isotope exchange study of GMPS (13).</p><p>Under conditions of low ATP concentration (up to two-fold molar excess of ATP over protein), the absorbance decays show a different behavior. Over the course of approximately one minute, the decay returns to the level seen at the start of the mixing (Figure 6). This is consistent with a slow hydrolysis of the adenylylate intermediate to XMP and AMP, a process reported earlier (13). The approximate rate of this hydrolysis, as determined by non-linear curve fitting with the single-exponential model above, appears to occur at nearly the same or at a somewhat faster rate than the k−3 reverse reaction (Table 1). This process is indicated by the khydrolysis step in Scheme 3.</p><!><p>In order to examine the intrinsic steps in the GMPS reaction in the presence of an ammonia source, stopped-flow mixing of glutamine with the enzyme, pre-mixed with an excess of both nucleotide substrates, was performed. The steps in this case, the binding and hydrolysis of glutamine and the transfer of the resulting ammonia, give rise to rapid absorbance changes. Under conditions where XMP substrate level is held in molar equivalence with protein, a rise in 290 nm absorbance is observed, followed by a plateau with no further change (Figure 7A). These absorbance changes are consistent with those expected based upon the respective extinction coefficients as stated above (Figure S1).</p><p>Figure 7B shows the same experiment, except with XMP in excess of protein. As before, the absorbance rise also initially occurs, but in this case the plateau phase is replaced with a linear decline, consistent with the steady-state conversion of XMP to GMP as exploited for a standard steady-state assay for the enzyme(39). A very brief lag phase, consistent with glutamine binding, precedes the rise phase in both cases. In the single-turnover case, an increase in enzyme used in the experiment led to a proportional increase in the amplitude of the rise phase, as determined by a single-exponential curvefit to this phase; however, the rate of the rise stayed within the range of 35 to 40 s−1, independent of protein concentration (Figure 7C). Taken together with the ultraviolet spectra of adenylylated XMP and GMP, these data are consistent with the glutamine-dependent absorbance rise being due to a burst of conversion of enzyme-bound adenylylated XMP intermediate to GMP.</p><p>A kinetic model taking these glutamine-dependent data into account is shown in Scheme 3. In this model, reversible glutamine binding precedes the catalytic step of the enzyme, here represented by k5, which is comprised also of the glutamine hydrolysis and ammonia transfer, which are not resolvable by these experiments. Finally, the scheme shows release of products, which regenerates the free enzyme.</p><!><p>As the steady state turnover number of E. coli GMPS at 25°C had not been previously reported, the value of this kinetic constant was determined in order to compare with the forward rates determined by pre-steady state methods. Two separate measurements of reaction velocity were made to arrive at the kcat for the enzyme at 25°C: a continuous assay monitoring steady-state conversion of XMP to GMP, and a glutamine hydrolysis assay, which quantitates production of glutamate. Both assays produced essentially identical kcat values of 6.9 ± 0.1 s−1 (nucleotide conversion) and 7.5 ± 0.1 s−1 (glutamate production).</p><!><p>Key themes in glutamine amidotransferase catalysis include the upregulation of the catalytic activity of the glutaminase domain by binding of an acceptor substrate in a separate synthetase domain, and intramolecular channeling of the glutamine-derived ammonia between distal active sites (43, 44). GMPS demonstrates this conditional glutaminase activity (45) and is also thought to create an ammonia channel via large-scale conformational changes (17, 31). Details on the kinetic and structural events related to channel formation and glutaminase function, however, have been lacking. A feature of GMPS function that proved useful in analyzing these details was the sequential and independent nature of the nucleotide activation and the glutamine utilization events enabling separate kinetic investigation of both processes with rapid reaction techniques.</p><p>In the kinetic mechanism presented here, a conformational change step follows the fast binding of XMP substrate. The occurrence of a conformational-response in GMPS upon substrate binding was noted in an earlier report, which indicated that the enzyme underwent a transition to a compact, protease-resistant structure in the presence of XMP, ATP, and Mg2+ (31). The substrate-induced intrinsic tryptophan fluorescence quenching reported here is consistent with this result and indicates that fast XMP binding is followed by a slower conformational change, estimated at 104 s−1. For the first time, the conformational transition of this amidotransferase can be correlated with a structural feature in the catalytic cycle. E. coli GMPS contains eight tryptophans scattered throughout its structure (46) (Figure S2). The judicious selection of site-directed tryptophan mutants established that the fluorescence transition can be attributed to a large extent to the single W37. Further inspection of the protein structural information regarding this unique position in the E. coli GMPS indicates a consistent hypothesis for how this position serves as a suitable reporter of at least a part of the conformational transition (Figures S2). Tryptophan 37 is a non-conserved residue found on the surface of the glutaminase domain of GMPS, near the hinge region of the glutaminase-synthetase interface. In the E. coli crystal structure (17), which is an open conformation of GMPS, the W37 side chain is seen to be in a T-shaped pi-stack with F49 (Figure S3). Upon substrate binding, alignment of the domains required to bring them in proximity for ammonia transfer requires rearrangement within the region of the W37. As a result of this motion, it is plausible that W37 is exposed to bulk solvent and the intrinsic fluorescence is quenched.</p><p>One aspect of the GMPS conformational changes that occur during the catalytic cycle is a large-scale loop disorder-to-order transition to form the complete nucleotide active site. In the E. coli GMPS crystal structure (17), a highly conserved, 22-residue loop was disordered. A crystal structure of human GMPS (47), demonstrated that this region is involved in nucleotide binding with the loop ordered and specific residues interacting with XMP. This region of the protein appears to be crucial for substrate specificity in N-type ATP-pyrophosphatase proteins. For example, in NH3-dependent NAD+ synthetase, a substrate–binding and specificity loop was ordered, juxtaposed to its adenylylated nucleotide intermediate (48). The structures of other family members, such as argininosuccinate synthetase (49) and β-lactam synthetase (50), also suggest that structurally analogous regions of these proteins are responsible for interaction with their respective substrates.</p><p>In GMPS, XMP appears to play a key role in initiating a series of conformational and catalytic events. Upon binding to the protein, the nucleotide induces the transition of the loop to an ordered state. The observation is also corroborated by the earlier proteolysis protection data, since the loop sequence contains three trypsin cleavage sites (31). Loop closure likely leads to the establishment of a complete binding site for XMP, and may also contribute to the formation of an intramolecular ammonia tunnel between the active sites, as in PRPP amidotransferase (51). The model from the single turnover studies is consistent with the combination of XMP binding and adenylylation being efficiently coupled to the transmission of the allosteric signal to the distal glutaminase active site.</p><p>The available protein crystal structures and the associated biochemical data from the results presented here offer a perspective for analysis of the GMPS active site. In Figure 8 are shown the ligand binding interactions with AMP and XMP in the human GMPS structure. This figure was modeled by aligning E. coli GMPS structure (1GPM), which has AMP and pyrophosphate bound in the active site, with the human GMPS structure (2VXO), which contains only XMP (Figure S4). The structure for the nucleotide complex indicates a relevant perspective, supported by the close proximity of the phosphate of AMP to the O2 of XMP (4.2Å) and to the oxygen of the resultant inorganic phosphate (3.4Å). These orientations and interatomic distances are consistent with the structure representing the product complex resulting from hydrolysis of the adenylylated-XMP.</p><p>In Figure 8, several key protein interactions with the nucleotides and inorganic pyrophosphate are represented and summarized in Tables S2 and S4. The numbering given here are those of the E. coli enzyme. The residues R308 and F315 reside in alpha helix 10, which has been previously described as part of the adenine-specific binding region (17), appear to orient the bound AMP. Residues D239 and S240 on alpha helix 7, a part of the P-Loop common to nucleotide-binding enzymes, function to orient the triphosphate group of ATP in the active site. Adjacent to the P-loop and interacting with D239 is K381, a hydrogen-bonding residue to the pyrophosphate in the crystal structure and previously proposed to assist in the orientation of the phosphates of ATP (17). Lysine 381 is also in close proximity to the synthetase domain's solvent-exposed surface that faces the glutaminase domain, suggesting a role for conferring to the glutaminase domain the formation of the O2-adenylyl XMP.</p><p>Interdomain residue contacts are expected to play a role in transmission of the allosteric signal to the glutaminase domain, as observed for IGP synthase (52). The GMPS conformational changes occurring during the catalytic cycle are interdomain motions that bring the glutaminase and synthetase domains in juxtaposition; a necessity for formation of an intramolecular ammonia path. Despite the human GMPS crystal structure demonstrating the ordered nucleotide-binding loop, no additional structural data have fully captured this path between the active sites, which is a key aspect of glutamine amidotransferases (30, 52). The fluorescence changes reported here suggest that the large-scale conformational changes required to achieve a closed, active enzyme are initiated by XMP binding and adenylylation.</p><p>The equilibrium for XMP activation lies far toward the adenylylated form of the substrate. Although this adenylylated intermediate was observed to hydrolyze upon drying, in agreement with an earlier report (1), it was stable enough in solution to permit, for the first time, collection of a UV spectrum. The intermediate's absorbance at 290 nm is significantly less than either XMP substrate or GMP product. Combined with the independent, sequential nature of the substrate activation and glutamine utilization reactions, this absorbance difference permitted the monitoring of chemical conversions of nucleotides on the enzyme. The maximal rate of XMP adenylylation by E. coli GMPS (40 s−1) is slower than the rate of XMP-induced conformational change by a factor of 2 to 3. This rate of adenylylation is similar to that seen in other enzymatic adenylylation reactions, for example in substrate activation in threonyl-tRNA synthetase (53) and tyrosyl-tRNA synthetase (54).</p><p>The rates of events that occur during glutamine utilization were observed by pre-loading the enzyme with the XMP-adenylylate and subsequent mixing with glutamine. The 38 s−1 absorbance increase is attributed to chemical conversion of adenylylated-XMP on the enzyme to GMP. This single-turnover absorbance at 290 nm absorbance change fits a single-exponential function but reports only on the amination of the adenylylated XMP to generate the GMP product. The rate is also similar to those observed in other enzymatic adenylylate turnover reactions, for example in tyrosyl-tRNA synthetase (55). However, all steps of glutamine binding, glutamine hydrolysis, and ammonia transfer are included. The lack of an observable time lag suggests that binding and cleavage of glutamine are very fast events with respect to all other steps. In addition, the transfer of ammonia likely occurs at a rate approaching that of free diffusion with precedent from previously characterized glutamine amidotransferase ammonia tunnels (51, 56, 57).</p><p>The forward rate constants leading up to and including GMP formation are faster than the steady-state turnover number for the enzyme, determined here to be 7 s−1 at 25°C. This fact is consistent with product release being the rate-controlling step in the overall catalytic cycle and consisting of a slow relaxation of the compact, product-bound conformation back to the open state of free enzyme. The rate of the ATP binding step was not investigated, for two reasons. First, E. coli intracellular concentrations of ATP are likely high enough (ca. 7 mM (58)) under physiological conditions that the binding equilibrium would always be very far toward the bound state. Second, an earlier report indicated that the conformational response of GMPS required the presence of both ATP and XMP nucleotides, while ATP binding alone was insufficient to achieve this response (31, 58), suggesting that the key changes required to activate the enzyme in the ammonia transfer process are elicited by XMP binding. Likewise, a full elaboration of the glutamine-dependent steps was not pursued, for a similar reason that intracellular glutamine concentrations in enteric bacteria are in the low millimolar range (e.g., (59)), a condition in which binding of this substrate is also not a rate-limiting process in vivo.</p><p>In summary, the results reported here provide insights into the detailed mechanism of the substrate-induced cascade that coordinates conformational changes and interdomain signaling in GMPS. The enzyme undergoes a substrate-induced conformational change to a compact state upon nucleotide binding and activation. These XMP-induced changes likely include a large-scale loop disorder to order transition, whole-domain motions, and the transfer of intramolecular signals that activate glutaminase activity in the second domain. The nature of these conformational changes and interdomain signals remains a subject of investigation and will offer important insights into the overall regulation of the catalytic and allosteric properties of N-type ATP-pyrophosphatases and Class I glutamine amidotransferases. These features will be ultimately critical to understanding the complex allosteric functions of the additional domain structure occurring in the human enzyme.</p><p>
        
          Equation 1
          
            
              y
              =
              −
              
                Ae
                
                  −
                  Rt
                
              
              +
              C
            
          
        
</p><p>
        
          Equation 2
          
            
              
                k
                app
              
              =
              
                k
                
                  −
                  2
                
              
              +
              
                
                  
                    k
                    
                      +
                      2
                    
                  
                
                
                  1
                  +
                  
                    
                      
                        K
                        a
                      
                    
                    
                      [
                      S
                      ]
                    
                  
                
              
            
          
        
</p>