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Originally published In Press as doi:10.1074/jbc.M412018200 on December 7, 2004

J. Biol. Chem., Vol. 280, Issue 8, 6416-6422, February 25, 2005
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Competitive Inhibitors of Mycobacterium tuberculosis Ribose-5-phosphate Isomerase B Reveal New Information about the Reaction Mechanism*

Annette K. Roos{ddagger}, Emmanuel Burgos§, Daniel J. Ericsson{ddagger}, Laurent Salmon§, and Sherry L. Mowbray¶||

From the {ddagger}Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, Uppsala SE-751 24, Sweden, the §Laboratoire de Chimie Bioorganique et Bioinorganique, CNRS-UMR 8124, Institut de Chimie Moléculaire et des Matériaux d'Orsay, Batiment 420, Université Paris-Sud XI, Orsay F-91405, France, and the Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Box 590, Uppsala SE-751 24, Sweden

Received for publication, October 22, 2004 , and in revised form, November 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribose-5-phosphate isomerase (Rpi), an important enzyme in the pentose phosphate pathway, catalyzes the interconversion of ribulose 5-phosphate and ribose 5-phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-D-erythronohydroxamic acid (Km 57 µM) and 4-phospho-D-erythronate (Ki 1.7 mM) refined to resolutions of 2.1 and 2.2 Å, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the ribose 5-phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-D-erythronate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ribose-5-phosphate isomerases (Rpi1; EC 5.3.1.6 [EC] ) catalyze the conversion of ribose 5-phosphate (R5P) to ribulose-5-phosphate (Ru5P) and vice versa (Fig. 1). The reaction is involved in the pentose phosphate pathway and in the Calvin cycle of plants. Mutant studies in Escherichia coli have shown that Rpi activity is important in bacterial growth (1). A recent medical study has further revealed that a deficiency in human Rpi causes destruction of myelin sheaths (leukoencephalopathy), which in turn leads to extensive brain abnormalities (2).



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  		FIG. 1.
Isomerization reaction and inhibitor structures. The isomerization step catalyzed by Rpis is shown at the top. The inhibitors used in this study, which are intended to mimic the 1,2-cis-enediolate high energy intermediate, are shown at the bottom. Carbon atoms are labeled on the R5P substrate, as well as the 4PEH inhibitor; note that the numbering of inhibitors is shifted by one carbon unit with respect to that of the substrates.

 
Two nonhomologous Rpis have been identified and designated as RpiA and RpiB. The former type is most common, occurring in all three kingdoms of life. RpiBs have so far only been found in the genomes of some bacteria and protozoa (3). Genes encoding both classes of enzymes are present in some bacterial species, including E. coli (4, 5). The two E. coli enzymes have been shown to have very different structures and active site residues, indicating that they represent an example of independent evolution (3, 6). We have more recently solved the structure of RpiB from Mycobacterium tuberculosis (MtRpiB) and demonstrated that it can catalyze the isomerization of R5P and Ru5P with an efficiency very similar to that of the E. coli RpiB, despite differences in the active site residues (7). As for most isomerases, the reaction catalyzed by Rpis is believed to proceed through an enediolate intermediate (Fig. 1). By comparisons of the two RpiB structures and by docking experiments, roles for the various residues in this class of enzyme were proposed (7). However, the catalytic mechanism could not actually be established from this information alone.

The present paper describes an investigation of the binding of MtRpiB to several inhibitors that mimic the high energy intermediate of the isomerization reaction (Fig. 1). X-ray structures for two of these inhibitors, 4-phospho-D-erythronohydroxamic acid (4PEH) and 4-phospho-D-erythronate (4PEA), in complex with MtRpiB are also presented. The combined data allow a much more complete understanding of the reaction mechanism of RpiBs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Purification—The MtRpiB protein was cloned, overexpressed, and purified as described previously (7) with the exception that the final purification step (size exclusion chromatography) was performed with a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 10 mM {beta}-mercaptoethanol, 0.1 mM EDTA. The purified protein was concentrated to 24 mg/ml and stored in the same buffer with 40 mM NaCl, at 4 °C or at –80 °C.

Kinetic Assays—The inhibitory capacity of five different reaction intermediate analogs (illustrated in Fig. 1) toward MtRpiB was tested using a spectrophotometric assay (8) where the conversion of R5P to Ru5P was monitored directly as a change in absorbance at 290 nm. The 1-ml reactions (in 50 mM Tris-HCl, pH 7.5) were followed for 5 min at room temperature (~22 °C) after the addition of 90 nM MtRpiB. Kinetic constants were obtained from Hanes-Woolf plots. Where possible, Ki values were estimated from a series of different R5P concentrations at three different inhibitor concentrations. A Km value for each inhibitor concentration was obtained and entered into a new plot of the observed Km versus inhibitor concentration. The x intercept, which is equal to –Ki, was determined by linear regression. For substances showing weak inhibition, only IC50 values could be acquired. These were measured using a constant substrate concentration of 5 mM, chosen close to the Km for maximum sensitivity.

Crystallization, Data Collection, and Data Processing and Refinement—The protein was cocrystallized with inhibitors by vapor diffusion in hanging drops including 1 µl of protein and 1 µl of reservoir solution at 20 °C. Inhibitor and protein solutions were mixed 15 min prior to setting up the crystallization drops. MtRpiB (17 mg/ml, 0.49 mM) with 3mM 4PEH was crystallized with 1.55 M ammonium phosphate and 0.1 M HEPES, pH 7.5. MtRpiB (12 mg/ml, 0.35 mM) with 50 mM 4PEA was crystallized with 1.65 M ammonium phosphate, 0.1 M MES, pH 6. Needle-like crystals of dimensions 0.2 x 0.05 x 0.05 mm3 appeared after ~2 weeks. Before flash cooling in liquid nitrogen the crystals were transferred to a cryoprotectant consisting of reservoir solution plus 25% glycerol. X-ray data were collected at 100 K (Oxford Cryosystems) at beamlines BM14 (4PEH data) and ID29 (4PEA data), both at ESRF, Grenoble. All data were indexed with MOSFLM (9) and processed in SCALA (10) via the CCP4 interface (11). Data collection statistics are summarized in Table I. Both crystals had C2 symmetry with unit cell dimensions nearly identical to those of the original phosphate complex crystals (7).


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  		TABLE I
Data collection and refinement statistics

The information in parentheses refers to the highest resolution shell.

 
The five molecules in the asymmetric unit of the original structure (PDB code 1USL [PDB] , with water and phosphate ligands removed) were allowed to move independently in rigid body refinement, after which a round of restrained refinement was performed with REFMAC5 (12). Inspection of the resulting maps at the graphics display indicated that ligands had bound in each case. After rebuilding the protein with O (13) and further refinement, the ligands were fit into the observed density in all five active sites of the asymmetric unit. Repeated rounds of refinement, rebuilding, and addition of waters with ARP/wARP (14) resulted in the final models described in Table I.

Structural Comparisons and Interpretations of Ligand Interactions— The two inhibitor-complex structures were compared with each other and with the previously published phosphate complex structure in O. This program was also used to dock the {beta}-furanose and open chain forms of R5P manually in the position of the 4PEH inhibitor, using the phosphate groups and the two carbon atoms adjacent to them as a guide. Figures were created with O, Molray (15), ChemDraw (CambridgeSoft Corp.), and Canvas (Deneba Systems, Inc.).

Deposition of Data—Atomic coordinates and structure factor data for the two complex structures have been deposited in the Protein Data Bank (16) with the entry codes 2BES [PDB] and 2BET.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Enzymatic Activity—The inhibition studies of 4PEH and 4PEA are illustrated in Fig. 2; binding data for these and other inhibitors are summarized in Table II. The results indicate that 4PEH binds ~30 times more tightly than 4PEA. 4-Phospho-D-erythronamide (4PEAm), 4-phospho-D-erythronhydrazide (4PEHz), and 4-phosphonomethyl-D-erythronate (4PMEA) showed still weaker inhibition, and so for these compounds only IC50 values were obtained (although a Ki for 4PMEA could be estimated from the IC50 and is included in Table II).



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  		FIG. 2.
Inhibition of MtRpiB by 4PEH and 4PEA. In A, experiments at various concentrations of 4PEH are shown; Hanes-Woolf plots are used to present the data. The concentrations of inhibitor and the apparent Km obtained from best fit to the lines were: {blacktriangleup}, 0.05 mM 4PEH, y = 43.9x + 177.7 -> Km = 4.05 mM; {blacksquare}, 0.01 mM 4PEH, y = 42.0x + 118.4 -> Km = 2.82 mM; {square}, 0 mM 4PEH, y = 41.8x + 87.9 -> Km = 2.10 mM. The average kcat for these three series is 58.9 ± 0.9 s–1. In B, the apparent Km is plotted against the 4PEH concentration to estimate Ki. In C, a similar series of experiments is illustrated for 4PEA: {blacktriangleup}, 4 mM 4PEA, y = 54.5x + 275.6 -> Km = 5.10 mM; {blacksquare}, 2 mM 4PEA, y = 51.5x + 150.8 -> K = 2.90 mM; {square}, 0 mM 4PEA, y = 47.4x + 72.7 -> Km = 1.54 mM. The average kcat for these three series is 49.4 ± 3.9 s–1. The apparent Km is plotted against the 4PEA concentration to estimate Ki in D.

 


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  		TABLE II
Inhibition data

IC50 values are given in parentheses. The IC50 values for MtRpiB with 4PEAm and 4PEHz and the Ki for 4PMEA are approximate. Data for the spinach enzyme are derived from previous work (23, 24). ND, not determined.

 
Complex Structures—The 4PEH complex structure was refined to 2.1 Å resolution and the 4PEA complex structure to 2.2 Å (see Table I). Both show density starting at residues 2–3 and ending at 158–160 (of 172, including the 10 residues of the His tag). MtRpiB forms a functional dimer as reported previously (7), with the two active sites of a dimer receiving contributions from both subunits (Fig. 3). Each asymmetric unit in the crystal contains two complete dimers; a fifth subunit is present which forms a dimer through crystallographic symmetry. Thus there are five independent observations of the MtRpiB active site for each complex structure determined. The electron density observed in the active sites of the 4PEH structure was consistently stronger than that for the 4PEA complex, although the density for the latter was clear (compare Fig. 4, A and B); this observation is in accordance with the tighter binding of the 4PEH ligand (Table II). Both complex structures were obtained in the presence of ~1.5 M phosphate, which has previously been shown to inhibit MtRpiB with a Ki of 130 mM (7). Clearly, the weaker affinity of the 4PEA ligand makes it more difficult for it to compete, even when it is included at 50 mM in the crystallization. Calculations suggest that a significant fraction (~30%) of the MtRpiB molecules is still bound to phosphate in the observed 4PEA structures. A mixed population is further suggested by the higher temperature factors of the ligands/protein in the 4PEA models (Table I), as well as by the slightly higher temperature factors (by ~5 Å2) for atoms at the nonphosphate end of the 4PEA ligands.



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  		FIG. 3.
Overall structure of the M. tuberculosis RpiB dimer. 4PEH ligands are shown bound in both active sites. The A molecule is colored in dark gray, the B molecule is pale gray, and 4PEH is black.

 



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  		FIG. 4.
Inhibitor binding to RpiB. Electron densities (using SIGMAA-weighted maps contoured at 1 {sigma}) for the two ligands, 4PEH (A) and 4PEA (B), are shown together with the residues of the active site believed to be important for enzymatic activity. The A molecule is a darker shade of gray than the B molecule. Water molecules are presented as small gray spheres. In C and D, the hydrogen bonding interactions observed in the active sites (cut-off distance 3.2 Å) are shown for the 4PEH and 4PEA complexes, respectively. The distances reported here from the A/B dimers are very similar to those observed in the other active sites.

 
Comparison of Complex Structures—When the A/B dimers of the two inhibitor complex structures are compared by superpositioning of C{alpha} atoms, it can be seen that these structures are very similar. The root mean square distance between them is 0.13 Å when 310 of the 314 C{alpha} atoms of the A subunits are compared, within the expected experimental error; the slight differences observed are restricted to the extreme N and C termini. Similar results are obtained when evaluating the other molecules in the asymmetric unit.

Comparing either of the two inhibitor complex structures with the original phosphate complex gives slightly higher root mean square distances, ~0.19 Å with ~300 C{alpha} atoms matching. The largest differences in these cases are seen in the A, C, and E molecules. Here, the N and C termini are seen to move slightly, and four loops close to the active site (generally 11–13, 41–44, 69, 111–112) are shifted by 0.4–0.7 Å, causing the active site residues to come closer to the bound inhibitor.

Ligand Interactions—Both 4PEA and 4PEH are bound in a similar fashion in the active site (Fig. 4). In each case, the phosphate group is placed at the same position as the phosphate in the original structure. In this location, it is largely exposed to the solvent; the other ends of the inhibitors point into a deep pocket in the enzyme. For one active site of the A/B dimer in the 4PEH complex, hydrogen bonds to the phosphate are contributed by residues His12 and Arg113 in the A molecule, and arginines 137 and 141 in the B molecule. Two water molecules are also in close proximity to the phosphate oxygens (one bound to Ala42 from the A molecule, and Arg141 from the B molecule, and one close to Arg113, A molecule). In the 4PEA complex structure the same residues interact with the phosphate in a very similar fashion (compare Fig. 4, C and D). The only (and small) difference is that Arg113 has a lower occupancy for this orientation and could be taking on an alternate conformation in some molecules of the asymmetric unit, as had been observed in the original phosphate complex structure. In the B, D, and E molecules this side chain clearly points toward the phosphate, but in the A and C molecules, the second conformation also appears to exist.

Moving deeper into the active site, the hydroxyl group on C3 of 4PEH (which corresponds to C4 of the open chain form of the substrate) interacts with His102 and a water molecule, which in turn is within hydrogen bonding distance of His138. In 4PEA the distance to His102 is longer (and presumably the interaction is weaker). The C2 hydroxyl group of 4PEH (corresponding to O3 of the substrate) interacts with the nitrogen backbone of Gly70 and the side chain of Asp11. In 4PEA, this same sugar hydroxyl group interacts with Asp11 and Glu75.

The terminal groups of 4PEH, which were designed to mimic the 1,2-cis-enediolate moiety of the intermediate of the isomerization reaction, have strong hydrogen bonds to the backbone nitrogens of residues 71 and 74. C1 of the substrate corresponds to the nitrogen atom of the hydroxamic moiety, which is situated 3.1 Å away from one carboxylate oxygen of Glu75. The other carboxylate oxygen of this same residue is close to both the amino group of the ligand and the hydroxyl group attached to it. The side chain of Ser71 is also seen to interact with the N-OH oxygen of 4PEH; it is slightly more distant (3.3 Å) from the O1 of 4PEH which replaces O2 of the substrate.

By contrast, there are few interactions between protein and the anionic group at the equivalent position in the 4PEA complex structure. Only one strong hydrogen bond is observed, that from one of the carboxylate oxygens to a water molecule. A longer hydrogen bond links this same atom to the backbone amide nitrogen of Ser71. Other distances to main chain nitrogens in the 70–74 loop region are longer, in the range of 3.4 Å. The Ser71 side chain is generally 3.4 Å from the OH group (although in the D and E molecules a better H-bond of ~3.1 Å is formed), and both Glu75 carboxylate oxygens are situated ~3.4 Å away. The latter is not surprising, given the likely repulsion of groups bearing the same charge. In two molecules of the asymmetric unit the amide nitrogen of the Asn103 side chain is drawn closer to one of the carboxylate oxygens.

Docking of Substrates—The {beta}-anomer of the furanose form of R5P was placed manually in the position of 4PEH, using the equivalent atoms of 4PEH as a guide (Fig. 5). This docking suggests that the furanose ring will be tilted toward His102 and His138, positioning the C1 hydroxyl group within hydrogen bonding distance of His138. In this way, the substrate is positioned correctly to use these histidines as catalysts for the ring opening step of the reaction. Docking of the {alpha}-anomer, on the other hand, brings the hydroxyl group close to Asn103 and is thus not consistent with this catalytic step. When the open chain form is docked in the same fashion, the O1 and O2 groups are placed close to the side chains of Ser71 and Glu75.



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  		FIG. 5.
Docking of substrate. A {beta}-furanose form of R5P was docked manually in the active site of MtRpiB using the equivalent atoms of 4PEH as a guide.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Isomerization of R5P to Ru5P probably requires the open chain form of the sugar. Although a mechanism can be envisioned in which both isomerization and ring opening of cyclic R5P occur in a concerted manner, leading directly to the enediolate intermediate species, we believe this is very unlikely because of the much lower acidity of the C2 hydrogen in cyclic compared with linear R5P. Kinetic studies of the unrelated spinach RpiA (17) suggested that this enzyme binds the furanose form and assists in its opening, a conclusion that was supported by structural studies combined with computer docking (7). Because the {alpha}- and {beta}-furanose forms are more plentiful in solution than the linear aldehyde (18), ring opening by the enzymes would have clear advantages for efficient processing of substrate, providing that this ring opening step is not rate-determining. Based on the results reported here, we propose that the fully conserved histidines 102 and 138 of MtRpiB are involved in this first step and that the true substrate of the enzyme is the {beta}-furanose, as illustrated in Fig. 6. The hydroxyl group on C3 of 4PEH, which corresponds to the cyclic O4 oxygen of the furanose ring, interacts with His102 and a water molecule (Fig. 4). A similar hypothesis was put forward for phosphoglucose isomerase (PGI), i.e. that the ring opening of 6-phospho-{beta}-D-fructofuranose is catalyzed by His388 (19). Further, the docking of a {beta}-furanose form of R5P in MtRpiB (Fig. 5) shows that O1 is placed ~3 Å from His138, in the same place that the water molecule is observed in the 4PEH structure. His138 is therefore an excellent candidate for the base that accepts a proton from O1. After the ring has been opened, a water molecule could quickly move into the position vacated by the sugar oxygen (as observed in the 4PEH structure). His138 could then use this water to return a proton to His102 in the last step of catalysis, so regenerating the active form of the enzyme. Docking the {alpha}-furanose form of R5P brings O1 close to Asn103, a residue that cannot act as a base, leading to the conclusion that the {beta}-furanose is a more likely substrate for the enzyme.



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  		FIG. 6.
Reaction mechanism. Proposed mechanism of the reversible isomerization reaction of R5P to Ru5P catalyzed by ribose-5-phosphate isomerase from M. tuberculosis.

 
The residues of the protein that catalyze isomerization are clearly identified by the hydroxamic moiety of the 4PEH inhibitor in which the two oxygen atoms are cis to each other. This group interacts mainly with an anion hole consisting of backbone amide nitrogens from residues 70 to 74, as well as the side chain of Glu75. Thus Glu75 is in an excellent position to act as the catalytic base that transfers a proton between C1 and C2 of the substrates R5P and Ru5P (Fig. 6). As reported previously for Glu165 in the triose-phosphate isomerase (TIM)-phosphoglycolohydroxamic acid complex structure (20), and in contrast to Glu357 in the PGI-5-phospho-D-arabinonohydroxamic acid structure (21), the plane of the carboxylate moiety of Glu75 is roughly perpendicular to the plane of the 4PEH hydroxamic function (Fig. 7). If the Ru5P substrate binds in the same way as 4PEH, the pro-R hydrogen on C1 would be transferred to the syn sp2 orbital of one of the carboxylate oxygens (O{epsilon}1orO{epsilon}2) of Glu75. The syn orbitals, which have been reported to be orders of magnitude more basic than the anti orbitals (22), are in the plane of the carboxylate group and point inward, whereas the anti orbitals point outward, as observed for the two corresponding TIM and PGI structures mentioned above. In the MtRpiB·4PEH structure, Ser71 interacts with the oxygen atoms of the 4PEH hydroxamic function through three hydrogen bonds. In addition, the oxygen atom of the side chain of Ser71 and the hydroxamic acid function are in the same plane, as observed previously for the nitrogen atom of His95 in the TIM-phosphoglycolohydroxamic acid complex and the water molecule Wat241 in the PGI-5-phospho-D-arabinonohydroxamic acid complex (Fig. 7). His95 of TIM has been implicated in the proton transfer between the oxygen atoms O1 and O2 of the high energy intermediate 1,2-cis-enediolate through its imidazole/imidazolate couple, and Wat241 through its H2O/OH– couple in PGI (21). The remarkable overlap of Ser71, His95, and Wat241 is depicted in Fig. 7 for the three corresponding structures. Therefore, we propose that Ser71 has a similar role through its Ser-OH/Ser-O– couple, i.e. that it catalyzes proton transfer between O1 and O2 of the enediolate high energy intermediate in MtRpiB (Fig. 6). This serine is highly conserved, or replaced with the similar threonine, in all RpiBs so far identified (3, 7), underlining its functional importance. The main chain nitrogen atoms in the 70–74 region form an anion hole that is expected to assist catalysis by stabilizing the transient negative charges during the reaction. In the fourth step of the reaction Glu75 donates a proton to the Re face of C1, thus creating the double bonded O2 of the ketose, Ru5P. His102 then donates its hydrogen to O4 but quickly recovers one from His138 via a water molecule, and the catalytic cycle is complete. In the reverse reaction, that converting Ru5P to R5P, the same steps will occur, with the exception that the ring opening step is not needed in this direction. In the E. coli RpiB, a change in the active site is observed, such that a cysteine residue will take on the role of Glu75. However, we anticipate that this enzyme will use the same reaction mechanism in other respects.



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  		FIG. 7.
Structural alignments to the MtRpiB·4PEH complex. MtRpiB·4PEH (gray), TIM·PGH (complex with phosphoglycolohydroxamic acid, PDB code 7TIM [PDB] , black) and PGI·5PAH (complex with 5-phospho-D-arabinonohydroxamic acid, PDB code 1KOJ [PDB] , white) were aligned, using the CONHOH function of each bound inhibitor. The superposition shows the similar orientation of their respective catalytic bases for the isomerization step, i.e. Ser71/Glu75 for RpiB, His95/Glu165 for TIM, and Wat241/Glu357 for PGI.

 
4PEA binds more weakly to MtRpiB (Table II), largely by virtue of a reduced number of hydrogen bonding interactions with the protein (Fig. 4). 4PEH was in fact designed to look more like the cis-enediolate intermediate than 4PEA and accordingly to be a better inhibitor of Rpi. Surprisingly, both of these compounds inhibit spinach RpiA equally well, with Ki values of ~30 µM at pH 7.5 (see Table II and Ref. 23). The similarity in their binding to RpiA could be explained by the presence of a lysine in the active site (Lys94 in the E. coli RpiA), which has been proposed to stabilize the high energy intermediate (6). No positively charged group is found in an equivalent position in RpiB, and therefore the negatively charged 4PEA is not a good inhibitor. More effective stabilization of the reaction intermediate via the lysine of RpiA could also account for the observed difference in catalytic rate of the two enzymes; the kcat of RpiA is ~2,100 s–1 (6), whereas the kcat of MtRpiB is only ~120 s–1 (7).

It was noted that there is no hydrogen bond between MtRpiB and the phosphate oxygen linked to C4 of the inhibitors. This observation led us to conclude that a replacement of this oxygen of the inhibitor by a methylene group, giving the hydrolytically stable analog, 4PMEA, should not significantly impair binding to the active site of the enzymes. As established previously for spinach RpiA (24), kinetic results show that 4PMEA inhibits MtRpiB almost as well as 4PEA (Ki of ~2 mM, see Table II).

As for PGI, where 5-phospho-D-arabinonamide and 5-phospho-D-arabinonhydrazide were reported to inhibit the enzyme poorly (25), compounds 4PEAm and 4PEHz are weak inhibitors of spinach RpiA and are even worse inhibitors of MtRpiB. Both 4PEH and 4PEHz appear to be good structural mimics of the 1,2-cis-enediolate intermediate, in contrast to 4PEA and 4PEAm, which are shorter by 1 unit. However, the pKa of a hydroxamic acid is about 9.5 versus more than 15 for a hydrazide, which makes the former group much more easily ionized. Therefore, our results suggest that both structural analogy and ionization of the small molecule are important for good inhibition.

In summary, kinetic and structural results now allow a good understanding of the mechanistic action of MtRpiB. Comparisons with the available data for an unrelated class of isomerases, the RpiAs, provide further insights into the observed differences, and similarities, in the behavior of those enzymes.


   FOOTNOTES
 
* This work was supported by the Swedish Research Council, the Swedish Foundation for Strategic Research, and European Commission SPINE Grant QLG2-CT-2002-00988 and X-TB Grant QLRT-2000-02018. 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.

The atomic coordinates and structure factors (codes 2BES and 2BET) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Back

|| To whom correspondence should be addressed: Dept. of Molecular Biology, SLU, BMC, Box 590, Uppsala SE-751 24, Sweden. Tel.: 46-18-471-4990; Fax: 46-18-536971; E-mail: mowbray{at}xray.bmc.uu.se.

1 The abbreviations used are: Rpi, ribose-5-phosphate isomerase; MES, 4-morpholineethanesulfonic acid; MtRpiB, RpiB from M. tuberculosis; 4PEA, 4-phospho-D-erythronate; 4PEAm, 4-phospho-D-erythronamide; 4PEH, 4-phospho-D-erythronohydroxamic acid; 4PEHz, 4-phospho-D-erythronhydrazide; PGI, phosphoglucose isomerase; 4PMEA, 4-phosphonomethyl-D-erythronate; R5P, ribose-5-phosphate; Ru5P, ribulose 5-phosphate; TIM, triose-phosphate isomerase; Wat, water. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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