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Originally published In Press as doi:10.1074/jbc.M409613200 on December 22, 2004

J. Biol. Chem., Vol. 280, Issue 9, 7861-7866, March 4, 2005
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The Trifunctional Sulfate-activating Complex (SAC) of Mycobacterium tuberculosis*

Meihao Sun{ddagger}, John L. Andreassi, II{ddagger}, Shuqing Liu{ddagger}, Rachel Pinto§, James A. Triccas§, and Thomas S. Leyh{ddagger}

From the {ddagger}Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461-1926 and §Mycobacterial Research Group, Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown NSW 2042, Australia

Received for publication, August 20, 2004 , and in revised form, December 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria, is comprised of three reactions: the synthesis of adenosine 5'-phosphosulfate (APS), the hydrolysis of GTP, and the 3'-phosphorylation of APS to produce 3'-phosphoadenosine 5'-phosphosulfate (PAPS), whose sulfuryl group is reduced or transferred to other metabolites. The entire sulfate activation pathway is organized into a single complex in Mycobacterium tuberculosis. Although present in many bacteria, these tripartite complexes have not been studied in detail. Initial rate characterization of the mycobacterial system reveals that it is poised for extremely efficient throughput: at saturating ATP, PAPS synthesis is 5800 times more efficient than APS synthesis. The APS kinase domain of the complex does not appear to form the covalent E·P intermediate observed in the closely related APS kinase from Escherichia coli. The stoichiometry of GTP hydrolysis and APS synthesis is 1:1, and the APS synthesis reaction is driven 1.1 x 106-fold further during GTP hydrolysis; the system harnesses the full chemical potential of the hydrolysis reaction to the synthesis of APS. A key energy-coupling step in the mechanism is a ligand-induced isomerization that enhances the affinity of GTP and commits APS synthesis and GTP hydrolysis to the completion of the catalytic cycle. Ligand-induced increases in guanine nucleotide affinity observed in the mycobacterial system suggest that it too undergoes the energy-coupling isomerization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The sulfate activation pathway in Mycobacterium tuberculosis is organized into a single complex that consists of three catalytic activities: an adenylyl-transferase (ATP sulfurylase), encoded by cysD, that catalyzes nucleophilic attack of sulfate at the {alpha}-phosphorous of ATP to produce adenosine 5'-phosphosulfate (APS);1 a GTPase, encoded by cysN (a member of the EF-Tu family) (1, 2), whose activity is linked to the kinetics and energetics of the ATP sulfurylase reaction; and APS kinase, located at the C terminus of the cysN subunit, that phosphorylates APS at the 3'-hydroxyl to produce 3'-phosphoadenosine 5'-phosphosulfate (PAPS) (Reactions 1, 2, 3, respectively).

(REACTION 1)

(REACTION 2)

(REACTION 3)
The M. tuberculosis and Escherichia coli cysD and cysN sequences share considerable similarity; however, the E. coli APS kinase is expressed as a separate polypeptide, rather than a CysN domain. The organism-dependent fusion of the early cysteine biosynthetic enzymes is particularly interesting, given that the E. coli ATP sulfurylase forms a tight, catalytically coupled complex with the last enzyme in the cysteine biosynthetic pathway, O-acetylserine sulfhydrylase (3). The self-organizing tendencies in the pathway hint at a yet higher order assembly of the cysteine biosynthetic enzymes. The bifunctional ATP sulfurylase from E. coli has been studied in some detail; however, the trifunctional complexes have not yet been mechanistically characterized. This study explores the mechanism of the sulfate-activating complex (SAC) from M. tuberculosis.

The World Health Organization estimates that M. tuberculosis now infects 30% of the world's population and that new infections are incurred at a rate of ~1 individual/s (4, 5). The organism is a serious threat to the welfare of humanity. It inhabits the macrophage phagosome, an organelle designed to degrade bacteria (6). Mycobacteria evade destruction in the phagosome by inhibiting fusion with lysosomes, which furnish the bactericides needed to kill the organism (6, 7). Once engulfed by the macrophage, mycobacteria encounter signals that cause it to adjust its mRNA and protein levels. Among the small group of proteins up-regulated in the macrophage environment are the sulfate-activating enzymes (7, 8). The link(s) between up-regulation of the sulfate activation pathway and the bacterium's ability to adapt to the phagosome environment is not fully understood; however, intriguing correlates have been established. Intracellular levels of mycothiol, the mycobacterial glutathione-surrogate, correlate with significant changes in the antibiotic resistance of the organism and its ability to survive an oxidative environment such as that presented by the phagosome (9, 10). Sulfolipids, which are sulfated using PAPS, appear to be found exclusively in pathogenic strains of mycobacterium (11, 12) and can inhibit phogosomelysosome fusion (13, 14). These tangible links between sulfur-containing metabolites and the viability and antibiotic resistance of M. tuberculosis suggest that sulfur metabolism may prove a fertile area of anti-tubercular research.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
EnzChek phosphate assay kit was obtained from Molecular Probes. mGMPPNP was obtained from Jena Biosciences. [35S]SO4/Na2SO4 was purchased from ICN. [{gamma}-33P]ATP was purchased from PerkinElmer Life Sciences. MgCl2, ATP, ADP, AMP, PPi, GTP, {beta}-NADH, NADP+, phosphoenol pyruvate (PEP), glucose, KOH, Na2SO4, EDTA, lysozyme, {beta}-mercaptoethanol, and dithiothreitol were the highest grade available from Sigma. Glycerol, KCl, streptomycin sulfate, (NH4)2SO4, and Hepes were acquired from Fisher Scientific. Lactate dehydrogenase (rabbit muscle), glucose-6-phosphate dehydrogenase (yeast), pyruvate kinase (rabbit muscle), hexokinase (yeast), inorganic pyrophosphatase (yeast), phenylmethylsulfonyl fluoride, and pepstatin were purchased from Roche Applied Science. PEI-F TLC sheets were purchased from EM Science. DNA restriction enzymes were acquired from New England Biolabs. Competent E. coli (BL21(DE3)) were purchased from Novagen, and Pfu Turbo polymerase was purchased from Stratagene. Sephadex G-25 and Q-Sepharose resins were obtained from Amersham Biosciences. PAP-agarose was purchase from Sigma. APS was synthesized and purified as described previously (3). PAPS was purchased from Prof. S. Singer (University of Dayton, Dayton, OH). Recombinant E. coli ATP sulfurylase was expressed and purified as described previously (15, 16). The yeast HAL2 (PAPS nucleotidase) bacterial expression plasmid, pETHAL2, was a generous gift from Prof. John D. York of Duke University Medical Center. Recombinant yeast HAL2 was expressed in BL21(DE3) cells and purified as described previously (17).

Purification of SAC—The recombinant mycobacterial sulfate-activating complex was expressed in BL21(DE3) and purified as described previously except for the addition of an anion exchange purification step (Q Sepharose Fast-Flow resin) after size exclusion chromatography (18). SAC eluted in a linear salt gradient (0–0.7 M KCl, 50 mM Hepes, 1.0 mM EDTA, pH/K+ = 8.0, 4 °C) at 0.30 M KCl. SAC purity was judged, using Coomassie Blue staining of SDS-PAGE gels, to be ≥95%.

Initial Rate Studies—Typically, the initial rate of reaction was determined at the 16 conditions defined by a 4 x 4 matrix of substrate concentrations in which each substrate was varied from ~0.2 to 5.0 times its Km. Rates were measured during the first ~7% of reaction. Triplicate rate measurements were averaged and then fit by the method of weighted least squares using the SEQUEN program (19). The conditions specific to the individual studies are described below. Due to the low turnover and high substrate affinities associated with the forward and reverse APS kinase reactions, it was not possible to perform experiments in which all of the APS and PAPS concentrations were in vast excess over that of the active site. Simulations at the lowest concentrations of these substrates used in the experiments predict that ~15% of the substrate is bound to the enzyme (binary and ternary complexes) during the measurements, which leads to an ~8% increase in Km and an ~2% decrease in kcat.

The ATP Sulfurylase Forward Reaction—The kinetic constants for ATP and SO4 were determined at a saturating concentration of GTP (1.0 mM, 83x Km). The data and experimental conditions are contained in Fig. 1A and the corresponding legend. The Km for GTP was determined in an initial rate experiment in which the concentration of GTP was varied from 0.2x to 5x Km at a near-saturating concentration of ATP and SO4. The pyruvate kinase/lactate dehydrogenase coupling system regenerates GTP from the GDP produced by the hydrolysis reaction and ATP from the ADP formed by the phosphorylation of APS. Hence, the overall reaction oxidizes two equivalents of NADH per molecule of APS and/or PPi formed, and the measured initial rates were halved in the analysis of the data. The GTPase assay conditions were as follows: SAC (1.2 µM), SO4 (4.0 mM, 10x Km), MgATP (1.0 mM, 28x Km), GTP (2.5, 3.8, 7.2, and 65 µM), Hepes/K+ (50 mM, pH 8.0), KCl (50 mM), MgCl2 ([nucleotide] + 1.0 mM), PEP (2.0 mM), NADH (0.3 mM), pyruvate kinase (10 units/ml), lactate dehydrogenase (20 units/ml), inorganic pyrophosphatase (1.0 unit/ml), and T = 25 ± 2 °C.



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  		FIG. 1.
Initial rate behavior of the M. tuberculosis SAC. A, initial rate study of the ATP sulfurylase-catalyzed APS synthesis reaction at a saturating concentration of GTP. The reaction conditions were as follows: SAC (1.5 µM), SO4 (75, 120, 250, and 2000 µM), ATP (17, 27, 54, and 500 µM), GTP (1.0 mM; 83x Km), Hepes (50 mM, pH/K+ = 8.0), KCl (50 mM), MgCl2 ([nucleotide] + 1.0 mM), PEP (2.0 mM), NADH (0.30 mM), pyruvate kinase (10 units/ml), lactate dehydrogenase (20 units/ml), inorganic pyrophosphatase (1.0 unit/ml), and T = 25 ± 2 °C. Triplicate measurements made at each substrate combination were averaged and fit to a sequential mechanism using the SEQUEN algorithm (19). Rates were measured during the first 7% of reaction. The solid lines represent the behavior predicted by the best-fit initial rate parameters (see Table I). B, double-reciprocal progress curves for the APS kinase-catalyzed synthesis of APS. The reaction conditions were as follows: SAC (8.0 µM, 14 milliunits/ml), PAPS (concentrations are indicated; see "Materials and Methods"), ADP (initial concentrations are indicated and increase during turnover; see "Results and Discussion"), hexokinase (14 units/ml), glucose-6-phosphate dehydrogenase (8.0 units/ml), E. coli ATP sulfurylase (1.0 units/ml), Hepes (50 mM, pH/K+ 8.0), glucose (2.0 mM), PPi (1.5 mM), NADP+ (0.20 mM), MgCl2 (1.0 mM + [ADP] + [PAPS] + [PPi]), and T = 25 ± 2 °C. Initial rates were calculated from progress curve tangents (see "Results and Discussion"). The data were fit to a rapid equilibrium model for a sequential mechanism using the SEQUEN algorithm. The solid lines represent the behavior predicted by the best-fit parameters (see Table I).

 
The ATP Sulfurylase Reverse Reaction—ATP synthesis was monitored continuously, using fluorescence spectroscopy ({lambda}ex = 339 nm, {lambda}em = 460 nm), by coupling its formation to the reduction of NADP+ using hexokinase and glucose-6-phosphate dehydrogenase. The assay conditions were as follows: SAC (2.0 µM), APS (5, 10, 29, and 90 µM), PPi (140, 240, and 700 µM), Hepes (50 mM, pH/K+ = 8.0), KCl (50 mM), MgCl2 ([nucleotide] + 1.0 mM), PEP (2.0 mM), NADP+ (0.25 mM), glucose (2.0 mM), hexokinase (36 units/ml), glucose-6-phosphate dehydrogenase (26 units/ml), and T = 25 ± 2 °C.

The APS Kinase Forward Reaction—Initial rates were measured in triplicate at the 16 conditions described by a 4 x 4 matrix of APS and ATP concentrations. Given the very low Km APS (0.30 ± 0.01 µM; Table I), the subsaturating APS concentrations needed to execute the initial rate titrations were too low to produce measurable quantities of product. To circumvent this problem, APS was regenerated from PAPS by the 3'-nucleotidase, HAL2 (17). ATP was regenerated from ADP using pyruvate kinase, and the reactions were continuously monitored at 339 nm by coupling the regeneration of ATP to the oxidation of NADH using lactate dehydrogenase. The assay conditions were as follows: SAC (0.05 µM), APS (64, 94, 177, and 1600 nM), ATP (16, 24, 45, and 400 µM), pyruvate kinase (10 units/ml), lactate dehydrogenase (10 units/ml), HAL2 nucleotidase (0.5 unit/ml), Hepes (50 mM, pH/K+ = 8.0), PEP (1.0 mM), NADH (0.25 mM), MgCl2 (2.0 mM), and T = 25 ± 2 °C.


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  		TABLE I
Initial rate kinetic constants for the SAC reactions

 
The APS Kinase Reverse Reaction—The data and experimental conditions associated with this experiment are contained in Fig. 1B and its legend. The progress curves were initiated at a near-saturating concentration of PAPS and contained varying initial ADP concentrations. APS was calculated using the change in absorbance at 365 nm ({epsilon}*365 nm = 6.82 mM–1 cm–1) associated with the production of 2 mol of ATP per mole of APS formed. The PAPS concentration was calculated by subtracting the concentration of APS formed at a given point in the reaction from the initial concentration of PAPS. ADP concentration was determined by adding the concentration of APS formed (which is converted to ADP by the coupling system) to the initial ADP concentration. The initial rates were calculated from the slopes of lines (–d[S]/dt) taken over intervals that spanned 1.5–2% of the overall reaction. The center points of the slopes were used to calculate the substrate concentrations. Typically, 200 V and [S] pairs were extracted from the 10,000–15,000 data points associated with a single progress curve, and kinetic parameters were obtained by statistically fitting the pairs to a model for a sequential reaction mechanism using the SEQUEN program (19).

The APS Kinase Forward Reaction using GTP—The initial rate measurements were performed at a fixed, saturating concentration of APS (50 µM, 330x Ki APS). GDP formation was monitored continuously at 339 nm using the enzymes pyruvate kinase and lactate dehydrogenase, which couple the regeneration of GTP to the oxidation of NADH. The assay conditions were as follows: SAC (0.75 µM), APS (50 µM), GTP (25, 50, 100, 200, and 500 µM; 0.3–6x Km app), pyruvate kinase (10 units/ml), lactate dehydrogenase (20 units/ml), Hepes (50 mM, pH/K+ = 8.0), PEP (1.0 mM), NADH (0.25 mM), MgCl2 ([nucleotide] + 1.0 mM), and T = 25 ± 2 °C.

The Native Molecular Mass of SAC—The molecular mass of SAC was determined by size exclusion chromatography using a Superdex 200 10/300 GL column (Amersham Biosciences) calibrated with molecular mass standards (ferritin, 440 kDa; catalase, 232 kDa; albumin, 67.0 kDa; ovalbumin, 43.0 kDa; and chymotrypsinogen A, 25.0 kDa). The standards yielded an excellent, linear standard curve when log molecular mass was plotted versus elution volume. The column, sample, and running buffer (50 mM Hepes/K+, 50 mM KCl, pH/K+ = 8.0) were equilibrated at 25 ± 2 °C. The apparent native molecular mass of SAC is 307 ± 7.0 kDa.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The Catalytic Behavior of the M. tuberculosis SAC—To define and compare the catalytic behavior of the M. tuberculosis SAC domains, their forward and reverse reactions were characterized using initial rate measurements. With the exception of the reverse APS kinase reaction, the experimental designs are classical (see "Materials and Methods"); a representative dataset is shown in Fig. 1A. The best-fit initial rate parameters obtained from these studies are compiled in Table I. The reverse APS kinase reaction is complicated by the well-established, potent inhibition by APS (20–22). ATP sulfurylase from E. coli, which does not have an APS kinase domain, can be used to remove APS and mitigate inhibition; however, this requires the presence of PPi and produces a second equivalent of ATP for each APS kinase turnover. The ATP is then converted by the coupling enzymes (hexokinase and glucose-6-phosphate dehydrogenase) into ADP, which is a substrate for the APS kinase reaction. Thus, the substrate concentration increases as the system turns over. If the ADP concentration is saturating at t = 0 of reaction, an increasing ADP concentration will not influence the reaction rate significantly, and linear 1/v versus 1/[PAPS] data are expected. Alternatively, if the ADP concentration is subsaturating, the velocity will increase until saturation is achieved, predicting a double-reciprocal plot that is non-linear at subsaturation and becomes linear as the enzyme nears saturation with ADP.

An initial rate, double-reciprocal progress curve of the APS-forming reaction catalyzed by the SAC APS kinase domain is shown in Fig. 1B. Both products of the reaction, APS and ATP, are removed by coupling enzymes, ATP sulfurylase from E. coli K-12 (present in excess over the SAC ATP sulfurylase domain) and hexokinase and glucose-6-phosphate dehydrogenase, which stoichiometrically couple NADP+ reduction to the ATP-to-ADP conversion (23). Initial rates are obtained from progress curve tangents calculated as slopes taken over suitably small regions of the curve (1–2%). The slope center points correspond to specific concentrations of PAPS and ADP that can be calculated from the optical density changes associated with NADP+ reduction (see "Materials and Methods"). The initial rate and concentration data extracted from the curves were fit, using the SEQUEN program of Cleland, and the best-fit constants (Table I) were used to simulate the data (Fig. 1B, solid lines).

Comparison of the kinetic constants of the SAC domains reveals that the system is beautifully poised to capture the APS released by the ATP sulfurylase domain at the APS kinase active site and produce PAPS. During initial rate turnover at saturating ATP, the catalytic efficiency of PAPS synthesis is 5800x that of APS synthesis, i.e. (V/Km APS)/(V/Km SO4). The affinity of APS for the active site of APS kinase is several hundred-fold greater than that for ATP sulfurylase, and PAPS is formed 6-fold faster than ATP. Thus, notwithstanding issues of competitive binding and inhibition, APS released by the ATP sulfurylase domain will kinetically partition almost exclusively toward PAPS synthesis. The efficiencies (V/Km APS) of APS kinases that are not attached to their ATP sulfurylase counterparts, E. coli (~108 M–1 s–1) (22) and Penicillium chrysogenum (1.4 x 107 M–1 s–1) (21), are substantially greater than that of the M. tuberculosis APS kinase domain (1.9 x 106 M–1 s–1). It may be that the efficiency demands placed on the co-localized systems, in which APS is released in the near vicinity of the APS kinase active site, are less than those placed on the systems that scavenge APS from the cellular milieu.

The SAC Quaternary Structure—The apparent native molecular mass of SAC, determined by size exclusion chromatography, is 307 ± 7.0 kDa (see "Materials and Methods"). The molecular masses of the SAC subunits, calculated from the translated DNA sequences, are 34,900 Da (CysD) and 67,800 Da (CysN/C); the mass of a single CysD·CysN/C heterodimer is 103,000 Da. The simplest interpretation of this data, which is supported by visual inspection of Coomassie Blue-stained SDS-PAGE gels, is that the native complex is a trimer of heterodimers (18); however, the presence or absence of a single additional CysD subunit in the complex cannot be ruled out. It is interesting to note that the tertiary structure of the M. tuberculosis system differs from that of E. coli, which is a tetramer of heterodimers.

The Absence of an E·P Intermediate—During its catalytic cycle, APS kinase from E. coli K-12 is phosphorylated at Ser109 (22, 24, 25). The {gamma}-phosphoryl group of ATP is transferred to and from this active site Ser (26) in a kinetically competent fashion. Labeling of the enzyme is stoichiometric with the active site, and E·P survives size exclusion chromatography and overnight storage without detectable loss of label or transfer function. APS kinase from P. chrysogenum shares a great deal of sequence similarity with the E. coli enzyme (46% identity, 63% similarity); yet mutagenic substitution of Ser107, the Penicillium homologue of Ser109 (E. coli), with alanine has little effect on catalysis (27). These and other results led to the conclusion that the Penicillium enzyme does not form the E·P intermediate. Thus, the field is left with the interesting conundrum of extremely similar enzymes that utilize very different catalytic mechanisms.

To further characterize the SAC complex and add to what is known about the isozyme-specific formation of the E·P intermediate, an attempt was made to phosphorylate the SAC APS kinase domain under conditions that stoichiometrically label APS kinase from E. coli. To ensure that the labeling conditions were reliable, the E. coli APS kinase was labeled (22) in a positive control experiment. The proteins were incubated with the labeling reagents and then separated from them using size exclusion chromatography. The protein concentration and E-33P profiles of the column eluants were constructed and compared (Fig. 2). The profiles associated with the E. coli labeling experiment were coincident, and the active site of the enzyme was 33P-labeled with ~96% efficiency. In contrast, 33P was not detected in the fractions containing SAC. The maximum level of labeled SAC that could have gone undetected in this experiment is <1% of an active site equivalent. The M. tuberculosis and E. coli systems clearly behave quite differently toward E·P formation, and it appears that, like its Penicillium counterpart, the M. tuberculosis system does not form the intermediate; however, E·P formation at very low levels cannot be ruled out by this experiment.



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  		FIG. 2.
Covalent labeling of SAC and the E. coli APS kinase with [{gamma}-33P]ATP. The labeling mixture contained E. coli APS kinase (50 µM) or SAC (33 µM), [{gamma}-33P]ATP (1.0 mM, specific activity = 239 nCi/µl), MgCl2 (2.0 mM), and Hepes (50 mM, pH 8.0/K+). The reactants were incubated together for 30 min at 25 ± 2 °C before loading onto a Sephadex G-25 column equilibrated and run at 4 °C with PO4 (50 mM), EDTA (2.0 mM), pH = 7.9. The protein concentration in each column fraction was measured by the method of Bradford (42), and 33P (in cpm) was determined by scintillation counting. The SAC data are represented by the solid symbols (A595, •; cpm, {blacktriangleup}); the E. coli data symbols are open (A595, {circ}; cpm, {square}).

 
The GTP Hydrolysis/APS Synthesis Stoichiometry—GTP hydrolysis is the only reaction of the SAC complex that produces Pi and can be monitored using a continuous Pi detection system (28). The presence of inorganic pyrophosphatase produces two equivalents of Pi from the PPi formed by the APS synthesis reaction. By comparing the initial rate of Pi formation in the presence and absence of inorganic pyrophosphatase, the stoichiometry of GTP hydrolysis and APS synthesis can be determined. This experiment was performed under the following conditions: SAC (0.44 µM), inorganic pyrophosphatase (0 or 3.2 units/ml), purine nucleoside phosphorylase (40 units/ml), ATP (1.0 mM, 29x Km), GTP (1.0 mM, 83x Km), Na2SO4 (7.6 mM, 19x Km), MgCl2 (3.0 mM), 2-amino-6-mercapto-7-methyl-purine riboside (0.40 mM), Hepes (50 mM), pH/K+ = 8.0, and T = 25 ± 2 °C. The ratio of turnover in the presence of inorganic pyrophosphatase (0.77 ± 0.01 s–1) to that in its absence (0.26 ± 0.01 s–1) is 3.0; hence, the stoichiometry of GTP hydrolysis to APS synthesis is 1:1. Whereas the results suggest that the two reactions are tightly coupled, they do not rule out the possibility of coupled and uncoupled reactions whose net behavior adventitiously produces a 1:1 stoichiometry.

The Chemical Potentials of GTP Hydrolysis and APS Synthesis Are Coupled—In the absence of GTP, APS synthesis is extremely unfavorable, Keq ~10–7 to 10–8 at near-physiological conditions (29). The SAC APS kinase domain couples APS synthesis to PAPS formation, and PAPS synthesis is sufficiently favorable (Keq = 2 x 103, 50 mM Hepes, pH/K+ = 8.0, T = 25 °C) (22) to produce detectable, albeit low, quantities of [35S]PAPS from ATP and 35SO4 in the absence of GTP; reactions initiated at 4.0 mM ATP and 0.50 mM 35SO4 produce a maximum of ~2% conversion of SO4 to PAPS, and APS levels under these conditions are too low to detect (see Fig. 3A). In the absence of GTP, the SAC concentrations needed for the PAPS synthesis reactions to reach completion within several hours are higher than the quantities of product formed. The 35SO4 assays measure the total product formed (i.e. enzyme-bound and solution-phase product); thus, it is possible that a significant fraction of the product is bound to SAC, a situation that prevents using the reaction end points to calculate solution-phase equilibrium constants. To assess whether the majority of the PAPS formed in the reaction is in solution, the progress curve was determined as a function of SAC concentration. SAC-bound PAPS will titrate with SAC concentration. If the SAC·PAPS complexes represent a significant fraction of the total PAPS formed at the reaction end point, the progress curve plateau will increase with increasing SAC concentration, which is not observed (Fig. 3A). Thus, the 9.5 µM PAPS that forms at the end point of the reactions is predominantly in solution, and one can calculate that the net equilibrium constant for conversion of ATP and SO4 to PAPS, ADP, and PPi is 4.4 x 10–4. Given Keq for the overall and PAPS-forming reactions, Keq for the APS synthesis reaction is calculated at 2.2 x 10–7, which corresponds to –9.0 kcal/mol, a number that agrees well with previously published data (30, 31). It is important to realize that APS formation is extremely unfavorable despite the fact that the {alpha},{beta}-bond of ATP is cleaved to produce it. {Delta}G'O for the hydrolysis of the {alpha},{beta}-bond, –10.4 kcal/mol, can be used in conjunction with the {Delta}GO for APS synthesis to estimate that {Delta}GO for the hydrolysis of the phosphoric-sulfuric acid anhydride bond of APS is a remarkably favorable –19.4 kcal/mol.



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  		FIG. 3.
The GTP hydrolysis and APS synthesis potentials are coupled. A, PAPS synthesis in the absence of GTP. The reaction solution contained SAC (15 µM, {circ}; 20 µM, {square}; 25 µM, •), ATP (4.0 mM), [35S]Na2SO4 (0.5 mM, specific activity = 100 nCi/µl), MgCl2 ([total nucleotide] + 1.0 mM), and Hepes (50 mM, pH 8.0/K+), T = 25 ± 2 °C. B, PAPS synthesis in the presence of GTP. The reactions contained SAC (10 µM; •) and GTP (3.0 mM; 250x Km); the remaining conditions are identical to those described in A. The low PAPS concentration dataset ({blacksquare}) is the same as that shown in A. The reactions were quenched at the indicated time intervals by the addition of EDTA (200 mM, pH 10.0) to a final concentration of 100 mM. Reactants were separated on PEI-F TLC sheets using a 0.90 M LiCl mobile phase and quantitated by two-dimensional imaging, as described previously (3). Each point is the average of two determinations.

 
The addition of GTP has a profound effect on the extent of SAC-catalyzed PAPS synthesis. A comparison of PAPS synthesis progress curves performed with and without GTP (at a saturating concentration) reveals that the PAPS formed at the end point of the (+)GTP reaction (470 µM) is 50-fold greater than that formed in its absence (see Fig. 3B). The apparent equilibrium constant for the APS-forming reaction in the presence of GTP is 0.23, which is comparable with the value of 0.059 determined using the E. coli system (32). The chemical potential of the hydrolysis reaction that is coupled to APS synthesis can be calculated from the difference in the potential of the APS-forming reaction in the presence and absence of GTP. This difference, –8.1 kcal/mol, agrees well with that expected for the hydrolysis of GTP (33). The thermodynamic and stoichiometry data both support the hypothesis that GTP hydrolysis and APS synthesis are tightly coupled and that the full complement of chemical potential available in the GTP hydrolysis reaction is used to drive the APS synthesis reaction forward.

The Isomerization of SAC—The mechanism of ATP sulfurylase from E. coli includes an isomerization that is driven by allosteric interactions between ligands at the GTPase and adenylyl-transferase active sites (16, 34). The isomerization, which precedes and partially rate-limits both GTP hydrolysis and APS synthesis, is a central energy-coupling step in the mechanism (34, 35). The isomerization commits the chemistries to forward reaction and appears to bring residues at both active sites into their catalytic positions, from which chemistry occurs quickly (35). Together, the substrate analogues AMP and PPi can substitute for ATP and SO4 in driving the isomerization and activating GTP hydrolysis (36).

The fluorescent, 3'-O-(N-methylanthraniloyl)-2'-deoxyguanine nucleotide derivatives (m-nucleotides) have proven valuable tools for understanding the allosteric interactions in the E. coli system and other GTPase-catalyzed reactions (37). These analogues are excellent functional mimics of their native counterparts and can be used to monitor nucleotide binding and hydrolysis. The quaternary GMPPNP·E·AMP·PPi complex of the E. coli system (and presumably the M. tuberculosis system) resembles a point in the native reaction in which the {alpha},{beta}-bond of ATP has been broken, and the {beta},{gamma}-bond of GTP has not. The system has isomerized and is stalled because it cannot cleave the {beta},{gamma}-imido bond of GMPPNP. Cleavage of the {beta},{gamma}-bond of GTP produces the GDP·E·AMP·PPi complex, which is not isomerized (34, 35). AMP and PPi are needed to experimentally observe the isomerization, and these activators increase the equilibrium of affinity of mGMPPNP for the E. coli enzyme 4700-fold (27 µM to 5.8 nM) (34).

To assess whether the M. tuberculosis complex undergoes an activator-dependent isomerization, the binding of mGMPPNP was studied in the presence and absence of saturating concentrations of AMP and PPi. The equilibrium binding of mGMPPNP to SAC is shown in Fig. 4A. The data are described well by a simple non-allosteric binding model with a Kd of 19 µM. The addition of AMP and PPi at saturating concentrations causes the affinity of mGMPPNP to increase 120-fold, to 160 nM (Fig. 4B). Thus, like its E. coli counterpart, SAC isomerizes in a ligand-dependent fashion in what is likely an energy-coupling step in the mechanism.



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  		FIG. 4.
Guanine nucleotide binding and isomerization of SAC. A, mGMPPNP binding in the absence of AMP and PPi. The titration was carried out by adding SAC to a solution containing mGMPPNP (0.50 µM), MgCl2 (1.0 mM), and Hepes (50 mM, pH/K+ = 8.0). The line through the data represents the best fit of the data using a non-cooperative binding model, Kd = 19 ± 1 µM. B, mGMPPNP binding in the presence of AMP and PPi. The titration was carried out by diluting a solution containing mGMPPNP (75 nM), AMP (1.0 mM), PPi (0.20 mM), MgCl2 (1.7 mM), Hepes (50 mM, pH/K+ = 8.0), and SAC (55 µM) with a solution that was identical, except that it did not contain SAC. The line through the data represents the best fit of the data using a non-cooperative binding model, Kd = 160 ± 20 nM. C, mGMPPNP binding in the presence of ATP. The titration was carried out by adding SAC to a solution containing mGMPPNP (1.0 µM), ATP (1.0 mM), MgCl2 (2.0 mM), and Hepes (50 mM, pH/K+ = 8.0). The line through the data represents the best fit of the data using a non-cooperative binding model, Kd = 2.3 ± 0.1 µM. D, mGMPPNP binding stoichiometry. The titration was carried out by adding SAC into a solution containing mGMPPNP (25 µM, ~11x Kd), ATP (1.0 mM), MgCl2 (2.0 mM), and Hepes (50 mM, pH/K+ = 8.0). The best-fit stoichiometry (1.1 ± 0.1) was obtained by fitting the data using a fixed Kd of 2.3 µM (see C). All of the experiments were performed at 25 ± 2 °C. The excitation and emission wavelengths were 354 and 445 nm, respectively. Each data point represents the average of at least two measurements.

 
The Stoichiometry of mGMPPNP Binding to SAC—The stoichiometry of mGMPPNP·SAC complex was determined in equilibrium titrations in which the mGMPPNP concentration was held fixed at 11x Kd mGMPPNP. Under these conditions, the majority of SAC remains bound to nucleotide until the concentration of SAC GMPPNP-binding sites begins to exceed that of the nucleotide, at which point the titration transitions into the plateau. The transition break point yields the stoichiometry. The titration was performed at a saturating concentration of ATP (1.0 mM) to make the experiments, which otherwise require very high concentrations of enzyme, more tractable by reducing the Kd of mGMPPNP (from 19 to 2.3 µM; see Fig. 4C). The ATP-dependent lowering of Kd underscores the reaction stage dependence of the allosteric communication between the active sites of the enzyme. The stoichiometry obtained by fitting the titration data (Fig. 4D) using a non-cooperative binding model and a Kd of 2.3 µM is 1.1 ± 0.1 mGMPPNP bound per GTPase subunit.

The ATP present in the stoichiometry titrations also prevents, by competitive binding, complications caused by mGMPPNP binding at the active site of the APS kinase domain. As is the case with APS kinase from other organisms, the SAC APS kinase domain is capable of using GTP as a substrate to phosphorylate APS (38–41). The kinetic constants associated with the GTP-dependent phosphorylation of APS are as follows: Km GTP = 90 ± 10 µM, and kcat = 17 ± 1 min–1 (see "Materials and Methods"). It appears that the metabolic links between sulfate activation and guanine nucleotides may extend beyond the coupling of GTP hydrolysis and APS synthesis.

Conclusion—The M. tuberculosis SAC has been physically and mechanistically characterized. The catalytic efficiency of the PAPS synthesis reaction is far greater than that of either of the reactions catalyzed by the ATP sulfurylase domain, a design that helps ensure that the system will produce PAPS efficiently in the presence of a metabolic demand. The complex appears to be organized as a trimer of heterodimers, ({alpha}{beta})3, which is distinctly different from the ({alpha}{beta})4 organization of the E. coli enzyme, which lacks the APS kinase domain. The stoichiometries of the APS synthesis and GTP hydrolysis reactions are, within error, identical, and the apparent equilibrium constant for APS synthesis increases 1.2 x 106-fold (–8.1 kcal/mol) during GTP hydrolysis; these facts reveal that the full chemical potential of the GTP hydrolysis reaction is harnessed to drive the synthesis of APS and that the energy-coupling efficiency of the enzyme is quite high. At the heart of the energy coupling lies a conformational change, induced by the binding of substrates or activators (AMP and PPi) that elicit an intermediate-like form of the enzyme, which increases the affinity of the enzyme for GTP and its analogues ~120-fold. The isomerization appears to require cleavage of the {alpha},{beta}-bond of ATP and commits the system to GTP hydrolysis and completion of the catalytic cycle; this interdigitation of the chemistries couples their chemical potentials. Equilibrium binding experiments using mGMPPNP show clearly that SAC undergoes this energy-coupling isomerization.

E. coli ATP sulfurylase forms a tight complex with O-acetylserine sulfhydrylase (3), the last enzyme in the cysteine biosynthetic pathway. New catalytic function emerges from the self-organization of these enzymes; the complex, not its constituents, can hydrolyze ATP. The hydrolysis of ATP is stoichiometric (1:1) with APS synthesis and is kinetically and energetically linked to turnover of ATP sulfurylase. Given the similarities between the E. coli and M. tuberculosis enzymes, it would not be surprising to discover that the mycobacterial system also forms a complex with other enzymes in the cysteine biosynthetic pathway and that that complex will exhibit interesting, linked catalytic functions.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM54469. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biochemistry, The Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461-1926. Tel.: 718-430-2857; Fax: 718-430-8565; E-mail: leyh{at}aecom.yu.edu.

1 The abbreviations used are: APS, adenosine 5'-phosphosulfate; mGMPPNP, 3'-O-(N-methylanthraniloyl)-2'-deoxy-{beta},{gamma}-imidoguanosine 5'-triphosphate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PEP, phosphoenol pyruvate; SAC, sulfate-activating complex. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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