Orientation of 1,3-Bisphosphoglycerate Analogs Bound to Phosphoglycerate Kinase*

David L. JakemanDagger §, Andrew J. IvoryDagger ||, G. Michael BlackburnDagger , and Michael P. Williamson§**

From the Dagger  Department of Chemistry and § Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom

Received for publication, November 19, 2002, and in revised form, December 23, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
THEORY
RESULTS
DISCUSSION
REFERENCES

We have previously reported dissociation constants for a range of bisphosphonate analogs of 1,3-bisphospho-D-glyceric acid binding to yeast phosphoglycerate kinase. Data for the unsymmetrical analogs were difficult to interpret because it was not clear in which of the two possible orientations these ligands bound. Here we report a novel NMR method for quantifying orientation preference based on relaxation effects induced by titration with CrADP, which is applied to these ligands. It is shown that all ligands can bind in both orientations but that the driving force for the orientational preference is to put the alpha ,alpha -difluoromethanephosphonate group in the "basic patch" (nontransferable phosphate) position. The relevance to the design of phosphoglycerate kinase inhibitors is discussed.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
THEORY
RESULTS
DISCUSSION
REFERENCES

Phosphoglycerate kinase (PGK)1 (EC 2.7.2.3) is a glycolytic enzyme that catalyzes the following reaction, where MgADP and MgATP represent the magnesium complexes of ADP and ATP, respectively.
1,3-<UP>Bisphosphoglycerate</UP><SUP>4−</SUP>+<UP>MgADP</UP><SUP>−</SUP>

 → 3-<UP>phosphoglycerate</UP><SUP>3−</SUP>+<UP>MgATP</UP><SUP>2−</SUP>

<UP><SC>Reaction</SC> 1</UP>
It is an attractive drug design target because trypanosomes rely on glycolysis as their sole source of energy, and hence inhibition of PGK could provide effective treatment for trypanosomal infections. The enzymes from a wide variety of sources have been compared (1). They show high sequence and structure similarity and consist of monomeric proteins of 45 kDa with two distinct domains connected by a hinge (Fig. 1). The N-terminal domain (which also contains the last 10 residues at the C terminus) has a "basic patch" containing a number of arginines and histidines that is the binding site for the glycolytic substrates 1,3-BPG and 3-PGA. The C-terminal domain is the binding site for ATP/ADP. The enzyme has a number of crystal structures (2-13). In many the two domains are in an open conformation with the substrates ~11 Å apart, which is much too far for direct phosphotransfer. Crystal structures also exist in which the enzyme forms a closed conformation with the two substrates close enough for direct nucleophilic associative phosphotransfer (14). A mechanism has been proposed based on these crystal structures of which the key features are that Arg-38 binds to the transferred 1-phosphate of 1,3-BPG and locates it close to the nucleophilic oxygen of ADP in the closed form, whereas Lys-213 (Lys-197 in Thermotoga maritima) (9) also binds to the phosphate, stabilizing the negative charge that develops on it as it transfers, thereby catalyzing the phosphoryl transfer. (Note: yeast PGK numbering has been used throughout.) The hinge bending has been studied in detail. Another subject of intense study is the kinetics of the reaction, which is activated by anions (15). Although the issue has not been resolved, it is likely that an anion binding site is formed when the two domains close (16).


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Fig. 1.   A schematic picture of the binding sites on PGK for 1,3-BPG and CrADP. No orientational or conformational detail is implied. In the closed conformation, the two domains come closer together such that the beta -phosphorus is close to the terminal phosphate of ADP.

The crystal structures have provided considerable information on how the substrates bind, both to the closed and to the open conformations. 1,3-BPG itself is very unstable, so no structures exist of bound 1,3-BPG. Crystals of 3-PGA show it bound with the 3-phosphate close to Arg-168, Arg-121, and Arg-65 in the basic patch, with the carboxylate close to Arg-38 and pointing toward the nucleotide. Somewhat surprisingly, in crystals of Trypanosoma brucei PGK grown in the presence of 3-PGA and MgADP from 2.5 M potassium phosphate, two of the four subunits in the asymmetric unit have a phosphate anion bound in the basic patch but close to Arg-38, i.e. close to the transferable phosphate site (11).

We have previously reported the synthesis and dissociation constants of PGK ligands based on 1,3-BPG (17, 18), which inhibit PGK (19). The phosphate linkages were replaced by non-scissile methanephosphonates, giving ligands that bind as much as 50 times more tightly than 3-PGA. It was observed that replacement of methanephosphonate by alpha ,alpha -difluoromethanephosphonate increased the affinity, and it was suggested that this was because of the lower pKa induced by the fluorines (18, 20). However, replacement of both methanephosphonates by difluoromethanephosphonates led to no further increase in affinity, suggesting that only one end of the molecule required the lower pKa and consequent greater charge density at neutral pH. Affinities could not be interpreted further because it was not possible to tell in which orientation the unsymmetrical ligands were binding. We have therefore developed a method to distinguish the orientation of the ligands and report its application with an analysis of the implications for design of PGK ligands.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
THEORY
RESULTS
DISCUSSION
REFERENCES

Yeast PGK was obtained from Roche Molecular Biochemicals as an ammonium sulfate suspension, resuspended in 0.1 M Tris, 0.1 mM EDTA, 6 mM 2-mercaptoethanol, 10 µM phenylmethylsulfonyl fluoride, and 5% ammonium sulfate, then concentrated into 40 mM KCl, 10 mM triethanolamine, pH 7.1 ± 0.05, in D2O. The protein concentration was typically 0.3 mM. PGK ligands were synthesized as described previously (18). CrADP was prepared as described (21).

One-dimensional 31P NMR spectra were acquired at 300 K on a Bruker AMX-500 spectrometer at 203.462 MHz using 60,000 scans over a spectral width of 8196 Hz and 2048 complex data points. A pulse angle of 60 ° (6.5 µs) was used with a relaxation delay of 0.5 s between pulses. Simultaneous decoupling of 1H and 19F was achieved during signal acquisition using a directional coupler. The probe was tuned to 19F, and optimal decoupling without disturbance of the signal was achieved using 1H decoupling (26 db attenuation of normal pulse power or ~330 Hz decoupling field at the sample) applied directly through the directional coupler and 19F decoupling (10 db attenuation or ~500-Hz field) into the side of the directional coupler. Both channels used Waltz-16 composite pulse decoupling. It proved particularly important to decouple the 31P signals efficiently from both 1H and 19F. In spectra where 19F was not decoupled, addition of CrADP gave rise to differential broadening of the 19F-coupled triplet with the outer lines broadening much more rapidly than the central line (22). This effect is caused by different relaxation rates of different elements of the scalar-coupled density matrix (23), possibly exacerbated by cross-correlation effects (24). Although these effects are of interest from a theoretical perspective, they make analysis of the relaxation data much more complicated. Indeed, simple analysis of the width of the central line gave results completely different from those obtained using complete decoupling. The free induction decays were zero filled to 4096 complex points, and then sensitivity enhanced by mild exponential multiplication of 2-3 Hz. A control spectrum was recorded with 1 molar eq of 1,3-BPG analog to PGK and no CrADP. Subsequent spectra were recorded after the addition of 12 mM CrADP. Three or four points were obtained for each analog with up to 1.5 eq of CrADP. The spectra were analyzed using Felix (Accelrys Inc., San Diego, CA) to measure the line width of the 31P signals by fitting to Lorentzian lines. The fitting was typically truncated at 10% of peak height. Errors were estimated by repeated fitting using different processing, base-line correction, and starting values for the fit. Simulations showed that the mild linebroadening applied had no significant effect on the line width ratios.

    THEORY
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The magnetic moment of an unpaired electron is approx 103 times greater than that of a nucleus. Fluctuation of local magnetic fields leads to relaxation, and the larger magnetic fields resulting from the presence of a paramagnetic species give rise to particularly efficient nuclear spin relaxation. The strong local magnetic field produced by an unpaired electron can be coupled to nuclei by dipole-dipole interactions. The dipolar effect of the unpaired electron on surrounding nuclei is inversely proportional to the sixth power of the distance between electron and nucleus, implying that the distance from a nucleus to an unpaired electron can be measured by its effect on nuclear transverse relaxation times (T2), which are proportional to line widths. In the experiments described here we measure the effect of Cr3+ on the relaxation of the two 31P nuclei in the ligand, here denoted alpha  and beta , and use the relative broadening to measure the distance to each nucleus and hence the orientation of the ligand.

Chromium(III) is a high spin (3d3 ion) dipolar relaxation probe. The contribution to the nuclear transverse relaxation rate (1/T2,p) from the paramagnetic probe is given by equations derived by Solomon and Bloembergen and presented by Mildvan and Gupta (Ref. 25 and references therein) as Equation 1.
<FR><NU>1</NU><DE>T<SUB>2,p</SUB></DE></FR>=fqF(&tgr;<SUB>c</SUB>)<FENCE><FR><NU>C</NU><DE>r<SUP>6</SUP></DE></FR></FENCE> (Eq. 1)
fq is the mole fraction of nuclei bound, C is a constant dependent on nuclear and electronic magnetic moments, r is the distance from Cr3+ to the relaxing nucleus, and F(tau c) is a function of tau c (correlation time for dipolar relaxation) and omega  (Larmor frequency of phosphorus). The line width at half-height (Delta nu 1/2,obs) in the presence of the paramagnetic ion is inversely proportional to the observed T2 (T2,obs, Equation 2).
&Dgr;&ngr;<SUB>1/2,<UP>obs</UP></SUB>=<FR><NU>2&pgr;</NU><DE>T<SUB>2,<UP>obs</UP></SUB></DE></FR> (Eq. 2)
Under conditions of fast exchange of the Cr3+ on and off the protein, the observed relaxation rate can be considered to arise from two contributions (Equation 3), namely, the relaxation rate in the absence of the paramagnetic ion 1/T2,abs, and the relaxation rate arising from dipolar interaction with the bound paramagnetic ion, 1/T2,p.
<FR><NU>1</NU><DE>T<SUB>2,<UP>obs</UP></SUB></DE></FR>=<FR><NU>1</NU><DE>T<SUB>2,<UP>abs</UP></SUB></DE></FR>+<FR><NU>1</NU><DE>T<SUB>2,<UP>p</UP></SUB></DE></FR> (Eq. 3)
Combining Equations 1, 2, and 3 for a 31P nucleus alpha  gives Equation 4 (tau c is the assumed constant).
&Dgr;&ngr;<SUB>1/2,<UP>obs</UP>,&agr;</SUB>=C′<SUB>&agr;</SUB>[<UP>CrADP</UP>]<FENCE><FR><NU>1</NU><DE>r<SUP>6</SUP><SUB>&agr;</SUB></DE></FR></FENCE>+k (Eq. 4)
k is a constant assuming all 1/T2,abs values are the same for all observed cases.

A plot of peak width at half-height versus [CrADP] has a gradient of C'alpha r<UP><SUB><IT>&agr;</IT></SUB><SUP>−6</SUP></UP>. Thus, a ratio of gradients allows us to estimate the relative distances of the 31P nuclei alpha  and beta  (ralpha /rbeta ) in the basic patch from the paramagnetic probe CrADP in the nucleotide binding site. The ratio of gradients is calculated according to Equation 5,
<FR><NU>gradient<SUB>&agr;</SUB></NU><DE>gradient<SUB>&bgr;</SUB></DE></FR>=<FR><NU>C′<SUB>&agr;</SUB>r<SUP>−6</SUP><SUB>&agr;</SUB></NU><DE>C′<SUB>&bgr;</SUB>r<SUP>−6</SUP><SUB>&bgr;</SUB></DE></FR>=<FENCE><FR><NU>r<SUB>&agr;</SUB></NU><DE>r<SUB>&bgr;</SUB></DE></FR></FENCE><SUP>−6</SUP> (Eq. 5)
because C'alpha  = C'beta . Thus, the 31P signal that broadens most on the addition of CrADP is the one closest to the Cr3+.

If the enzyme remains in its open conformation when the ligands bind, then the ratio ralpha /rbeta is fixed and can be estimated from crystal structures (3) as 1.8. Any ratio of gradientalpha /gradientbeta less extreme than 1/(ralpha /rbeta )6 = 1:34 can therefore be interpreted as caused by the ligand binding in both possible orientations. If the ligand binds in conformation I with probability p and in conformation II with probability 1 - p (Fig. 2), then the results are determined by Equations 6 and 7.
<FR><NU>1</NU><DE>T<SUB>2,p,<UP>I</UP></SUB></DE></FR> ∝ <FENCE><FR><NU>p</NU><DE>r<SUP>6</SUP><SUB>&agr;</SUB></DE></FR></FENCE>+<FENCE><FR><NU>(1−p)</NU><DE>r<SUP>6</SUP><SUB>&bgr;</SUB></DE></FR></FENCE> (Eq. 6)

<FR><NU>1</NU><DE>T<SUB>2,p,II</SUB></DE></FR> ∝ <FENCE><FR><NU>(1−p)</NU><DE>r<SUP>6</SUP><SUB>&agr;</SUB></DE></FR></FENCE>+<FENCE><FR><NU>p</NU><DE>r<SUP>6</SUP><SUB>&bgr;</SUB></DE></FR></FENCE> (Eq. 7)
Substituting Equations 6 and 7 into Equations 2 and 3, we obtain Equation 8.
<FR><NU>gradient<SUB>&agr;</SUB></NU><DE>gradient<SUB>&bgr;</SUB></DE></FR>=<FR><NU>C′<SUB>&agr;</SUB><FENCE><FENCE><FR><NU>p</NU><DE>r<SUP>6</SUP><SUB>&agr;</SUB></DE></FR></FENCE>+<FENCE><FR><NU>(1−p)</NU><DE>r<SUP>6</SUP><SUB>&bgr;</SUB></DE></FR></FENCE></FENCE></NU><DE>C′<SUB>&bgr;</SUB><FENCE><FENCE><FR><NU>p</NU><DE>r<SUP>6</SUP><SUB>&bgr;</SUB></DE></FR></FENCE>+<FENCE><FR><NU>(1−p)</NU><DE>r<SUP>6</SUP><SUB>&agr;</SUB></DE></FR></FENCE></FENCE></DE></FR>=<FR><NU>C′<SUB>&agr;</SUB><FENCE><FENCE><FR><NU>p</NU><DE>34r<SUP>6</SUP><SUB>&bgr;</SUB></DE></FR></FENCE>+<FENCE><FR><NU>(1−p)</NU><DE>r<SUP>6</SUP><SUB>&bgr;</SUB></DE></FR></FENCE></FENCE></NU><DE>C′<SUB>&bgr;</SUB><FENCE><FENCE><FR><NU>p</NU><DE>r<SUP>6</SUP><SUB>&bgr;</SUB></DE></FR></FENCE>+<FENCE><FR><NU>(1−p)</NU><DE>34r<SUP>6</SUP><SUB>&bgr;</SUB></DE></FR></FENCE></FENCE></DE></FR> (Eq. 8)
After rearrangement and using Equation 5, the apparent distance ratio is given by Equation 9, thereby allowing us to estimate conformational preference.
r<SUB>&agr;</SUB>/r<SUB>&bgr;</SUB>=<SUP>6</SUP><RAD><RCD><FENCE><FR><NU>34p+(1−p)</NU><DE>p+34(1−p)</DE></FR></FENCE></RCD></RAD> (Eq. 9)
This function is shown in Fig. 3, which shows that relatively small proportions of binding in the alternative conformation drastically reduce the apparent distance ratio.


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Fig. 2.   Two orientations with populations p and 1 -p.


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Fig. 3.   Plot of Equation 9.

If the enzyme conformation is altered in the direction of the closed form when the ligands bind, then both ralpha and rbeta are reduced and the ratio ralpha /rbeta increases. The conformational preference derived above (Equation 9) is therefore a maximum limit, and any closed form population brings the conformational preference closer to 50%.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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RESULTS
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REFERENCES

31P NMR spectra were acquired for a range of PGK ligands in 1:1 complexes with PGK, and line widths were measured as a function of added CrADP. The addition of CrADP caused a monotonic increase in line width as expected. From the 1H spectra acquired on titration of the ligands with PGK, it is clear that no large conformational changes occur in PGK, and thus the enzyme is expected to remain in the "open" conformation seen in the majority of crystal structures. Typical spectra are shown in Figs. 4 and 5 for a ligand containing only methanephosphonates and a ligand containing both a methanephosphonate and an alpha ,alpha -difluoromethanephosphonate, respectively. Differential broadening exists of the two 31P signals, which indicates a conformational preference of the ligand. Line widths were measured by line fitting, and the gradients of line width versus CrADP were compared to derive an effective distance ratio of the two phosphorus atoms from the chromium, as in Equation 5. A typical result is shown in Fig. 6 for compound 6 (data from Fig. 5). The results are summarized in Table II, which also presents the affinities measured previously. None of the distance ratios measured approached the theoretical limit of 1.8 as presented above (assuming that the open enzyme conformation is the same as that in the crystal). The largest ratio found was 1.49. We therefore used the distance ratios to calculate the approximate conformational preference of the ligands, which are listed in Table II. Conformational preferences range between 29 and 94%, with some analogs showing almost no orientational preference. We note that nonspecific binding of the ligand to PGK would reduce the ratio toward a 50% orientational preference; therefore, the values given in Table II represent minimum values for the conformational preference.


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Fig. 4.   CrADP titration of 1. The left-hand peak is the peak labeled alpha  in Table I. Protein and 1 concentration is 300 µM. The spectra represent no CrADP (a), 28 µM CrADP (b); 56 µM CrADP (c).


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Fig. 5.   CrADP titration of 6. The left-hand peak is the peak labeled alpha  in Table I. Protein and (6) concentration is 300 µM. The spectra represent no CrADP (a), 28 µM CrADP (b); 56 µM CrADP (c).


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Fig. 6.   Line widths measured for the CrADP titration of 6. Data are from Fig. 5. Triangles, Palpha ; squares, Pbeta . The lines are best fit lines to the data, with gradients of 1.2 and 0.58, respectively. The ratio ralpha /rbeta is then obtained as (1.2/0.58)-1/6 = 0.89.

The ligands were designed to have a similar electronic distribution to 1,3-BPG and 3-PGA in having either a simple ketone or an amide group in analogous positions to the substrates (Table I). Therefore it was expected that the non-symmetrical ligands would bind such that the carbonyl oxygen would occupy a location similar to that of the carboxylate of 3-PGA. The results presented in Table II for the nonfluorinated analogs 1, 4, and 5 show that they do indeed bind preferentially in the expected orientation but that the conformational preference is not strong.


                              
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Table I
Structures of analogs used in Cr3+ NMR experiments


                              
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Table II
Relative Cr3+ to 31P distances measured using paramagnetic titrations

We have previously shown that substitution of the methanephosphonate by the alpha ,alpha -difluoromethanephosphonate group almost always gave a large increase in affinity for PGK (18). (One of the few exceptions is 7, which is mainly in the diol form and therefore has steric problems in binding.) This was true whether the fluorines were placed at what was expected to be the 1-phosphate end (2) or the 3-phosphate end (3), yet substitution at both ends gave binding that was no stronger and indeed often weaker. The results (Table II) indicate that in all cases the difluoromethanephosphonate group is positioned at the 3-phosphate end. This finding makes sense of the affinities as discussed below.

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A quantitative use of paramagnetic relaxation as done here is only applicable if certain conditions are met. Many of the NMR-specific requirements have been addressed by Gupta and co-workers (25, 26), who showed in a study of pyruvate kinase that CrADP can be used as a substitution-inert paramagnetic analog of MgADP and that the relaxation effects are dominated by the paramagnetism of the chromium in a simple r-6 relationship. CrATP has been shown to be a suitable analog of MgATP when binding PGK, and its effects on longitudinal relaxation rates were used to probe substrate binding (27). It is also of course necessary that CrADP binds at a single site on PGK. In this case chromium is in a very similar position to magnesium (28, 29). Considerable debate has occurred as to whether 1,3-BPG and its analogs bind at more than one site on PGK (16), and it is now reasonably clear that at least in the open form it only has a single major binding site (13). Finally, by using a low concentration of both enzyme and CrADP along with tight binding PGK ligands we can ensure that essentially all of the paramagnetic relaxation of the ligand occurs while bound to the enzyme, and no significant relaxation occurs in a CrADP·ligand binary complex. This fact has been confirmed by measurements in the absence of enzyme (data not shown).

Apparent 31P-chromium distance ratios were obtained from measuring the change in line width of the 31P signal with the addition of CrADP. In all cases the ratios were substantially smaller than the theoretical limit of 1.8. This result could in principle be explained if the geometry of the bound 1,3-BPG analogs relative to CrADP is very different from that of 1,3-BPG so that the 31P-chromium distance ratios become more similar. This circumstance is unlikely. In PGK crystal structures (2-13) and in a modeling study (30) the only significant change is a complete or partial closure of the hinge between the two domains that brings the transferable 1-phosphate closer to the bound nucleotide while maintaining the same orientation and therefore makes the ratio ralpha /rbeta greater not smaller. The two most likely explanations for the reduced distance ratios are either that CrADP or the analogs bind weakly in different binding sites or that the 1,3-BPG analogs can bind in the correct binding site but with both possible orientations. The first option can be ruled out as discussed above. Thus, binding in two possible orientations is the most likely explanation for the low ralpha /rbeta ratio.

Equation 9 provides a crude but useful characterization of the approximate conformational preference of the 1,3-BPG analogs tested. The most striking result is that all of the alpha ,alpha -difluoromethanephosphonate analogs bind with the more highly charged difluoromethanephosphonate at the 3-phosphate end. This finding is perhaps surprising in view of the location of the phosphate anion at the 1-phosphate end in the T. brucei crystal structure, but it allows us to interpret the ligand affinities in a straightforward manner. We assume that the greater charge density induced by the difluoro substitution is the main driving force for the conformational preference seen. It may also be the case, however, that the fluorines can act as mimics of the lone pairs of the phosphate-bridging oxygen of the substrate and participate in hydrogen bonds to the enzyme, as seen in a crystal structure of glycerol phosphate bound to AMP-PCF2P (31). However, the result does agree with the long standing findings that the 3-phosphate groups should be doubly ionized to bind effectively (32) and that the binding of the carboxylate of 3-PGA is less important (33). We note that the strongest conformational preference was for 3, which has the amide functionality in a "substrate-like" location.

The results indicate that the location of the difluoromethanephosphonate dominates the orientation of binding and that any functionalization of the carbon chain between the phosphorus atoms has only a secondary effect. Thus, a difluoromethanephosphonate group at the 3-phosphate end is always favorable and will make the ligand bind in this orientation even if this means disrupting other substrate-like interactions elsewhere. The effect can be seen from a comparison of 2 and 3, both of which bind with the fluorines at the 3-phosphate end, despite putting the carbonyl group of 2 in the "incorrect" location. However, such "incorrectness" only reduces the affinity from 2 µM (3) to 6 µM (2). This conclusion makes sense of our previous study (18), which showed that substitution by a single difluoromethanephosphonate always improved affinity but that substitution of difluoromethanephosphonates at both ends of the ligands conferred no additional affinity and often reduced the affinity. We infer that the presence of a difluoromethanephosphonate group at the transferred 1-phosphate position must be unfavorable either for steric reasons or because the greater charge density on the difluoromethanephosphonate cannot easily be stabilized in the open form of the enzyme. The enzyme functions by stabilizing the developing negative charge on the transferring phosphate in the closed form (11), and therefore we suggest that inhibitors should only have a charge of -1 at the 1-phosphate end but a charge of -2 at the 3-phosphate end.

Our results suggest that the basic patch is important mainly as a region of high positive charge to locate the 3-phosphate of the substrate (34). This finding may explain why mutations in the basic patch have relatively little effect on the kinetics of the reaction (1, 35).

In summary, we have shown that the results obtained on the orientation of PGK ligands allow us to interpret the affinities measured previously. We suggest that an effective PGK inhibitor should have a high charge density at the non-transferred 3-phosphate end but a lower charge density at the transferred 1-phosphate end. Steric restrictions at the 3-phosphate end are not strong. We have not identified strong requirements for functionality within the ligand, although substrate-like functionality provides weak additional affinity.

    FOOTNOTES

* This work was supported by a studentship from the Biotechnology and Biological Sciences Research Council (BBSRC) (to D. L. J.) and BBSRC Grant GR/H/32780. The Krebs Institute is a BBSRC Biomolecular Sciences Centre.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: College of Pharmacy, 5968 College St., Dalhousie University, Halifax, Nova Scotia B3H 3J5, Canada.

|| Current address: Environment Australia, GPO Box 787, Canberra ACT 2601 Australia.

** To whom correspondence should be addressed: Dept. of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom. Tel.: 44-114-222-4224; Fax: -44-114-272-8697; E-mail: m.williamson@sheffield.ac.uk.

Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211769200

    ABBREVIATIONS

The abbreviations used are: PGK, yeast phosphoglycerate kinase; 1, 3-BPG, 1,3-bisphosphoglycerate; 3-PGA, 3-phosphoglyceric acid; AMP-PCF2P, beta ,gamma -difluoromethyleneadenosine 5'-triphosphate; mgATP, magnesium complex of ATP.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
THEORY
RESULTS
DISCUSSION
REFERENCES

1. João, H. C., and Williams, R. J. P. (1993) Eur. J. Biochem. 216, 1-18[Medline] [Order article via Infotrieve]
2. Banks, R. D., Blake, C. C. F., Evans, P. R., Haser, R., Rice, D. W., Hardy, G. W., Merrett, M., and Phillips, A. W. (1979) Nature 279, 773-777[Medline] [Order article via Infotrieve]
3. Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kingsman, A. J., and Kingsman, S. M. (1982) EMBO J. 1, 1635[Medline] [Order article via Infotrieve]
4. Harlos, K., Vas, M., and Blake, C. C. F. (1992) Proteins 12, 133-144[Medline] [Order article via Infotrieve]
5. Davies, G. J., Gamblin, S. J., Littlechild, J. A., and Watson, H. C. (1993) Proteins 15, 283-289[Medline] [Order article via Infotrieve]
6. Davies, G. J., Gamblin, S. J., Littlechild, J. A., Dauter, Z., Wilson, K. S., and Watson, H. C. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 202-209[CrossRef][Medline] [Order article via Infotrieve]
7. May, A., Vas, M., Harlos, K., and Blake, C. (1996) Proteins 24, 292-303[CrossRef][Medline] [Order article via Infotrieve]
8. McPhillips, T. M., Hsu, B. T., Sherman, M. A., Mas, M. T., and Rees, D. M. (1996) Biochemistry 35, 4118-4127[CrossRef][Medline] [Order article via Infotrieve]
9. Auerbach, G., Huber, R., Grättinger, M., Zaiss, K., Schurig, H., Jaenicke, R., and Jacob, U. (1997) Structure 5, 1475-1483[Medline] [Order article via Infotrieve]
10. Bernstein, B. E., Michels, P. A. M., and Hol, W. G. J. (1997) Nature 385, 275-278[CrossRef][Medline] [Order article via Infotrieve]
11. Bernstein, B. E., and Hol, W. G. J. (1998) Biochemistry 37, 4429-4436[CrossRef][Medline] [Order article via Infotrieve]
12. Bernstein, B. E., Williams, D. M., Bressi, J. C., Kuhn, P., Gelb, M. H., Blackburn, G. M., and Hol, W. G. J. (1998) J. Mol. Biol. 279, 1137-1148[CrossRef][Medline] [Order article via Infotrieve]
13. Szilágyi, A. N., Ghosh, M., Garman, E., and Vas, M. (2001) J. Mol. Biol. 306, 499-511[CrossRef][Medline] [Order article via Infotrieve]
14. Knowles, J. R. (1980) Annu. Rev. Biochem. 49, 877-919[CrossRef][Medline] [Order article via Infotrieve]
15. Scopes, R. K. (1978) Eur. J. Biochem. 91, 119-129[Abstract]
16. Szilágyi, A. N., and Vas, M. (1998) Biochemistry 37, 8551-8563[CrossRef][Medline] [Order article via Infotrieve]
17. Blackburn, G. M., Jakeman, D. L., Ivory, A. J., and Williamson, M. P. (1994) Bioorg. Med. Chem. Lett. 4, 2573-2578[CrossRef]
18. Jakeman, D. L., Ivory, A. J., Williamson, M. P., and Blackburn, G. M. (1998) J. Med. Chem. 41, 4439-4452[CrossRef][Medline] [Order article via Infotrieve]
19. McHarg, J., and Littlechild, J. A. (1996) J. Pharm. Pharmacol. 48, 201-205[Medline] [Order article via Infotrieve]
20. Caplan, N. A., Pogson, C. I., Hayes, D. J., and Blackburn, G. M. (2000) J. Chem. Soc. Perkin Trans. I, 421-437
21. Dunaway-Mariano, D., and Cleland, W. W. (1980) Biochemistry 19, 1496-1505[Medline] [Order article via Infotrieve]
22. Bendel, P., and James, T. L. (1982) J. Magn. Reson. 48, 76-85
23. Vold, R. R., and Vold, R. L. (1976) J. Chem. Phys. 64, 320-332
24. Werbelow, L. G., and Grant, D. M. (1977) Adv. Magn. Reson. 9, 189-299
25. Mildvan, A. S., and Gupta, R. K. (1978) Methods Enzymol. 49, 322-359[Medline] [Order article via Infotrieve]
26. Gupta, R. K., and Benovic, J. L. (1978) J. Biol. Chem. 253, 8878-8886[Medline] [Order article via Infotrieve]
27. Gregory, J. D., and Serpersu, E. H. (1993) J. Biol. Chem. 268, 3880-3888[Abstract/Free Full Text]
28. Tanswell, P., Westhead, E. W., and Williams, R. J. P. (1976) Eur. J. Biochem. 63, 249-262[Abstract]
29. Lester, L. M., Rusch, L. A., Robinson, G. J., and Speckhard, D. C. (1998) Biochemistry 37, 5349-5355[CrossRef][Medline] [Order article via Infotrieve]
30. Chandra, N. R., Muihead, H., Holbrook, J. J., Bernstein, B. E., Hol, W. G. J., and Sessions, R. B. (1998) Proteins Struct. Funct. Genet. 30, 372-380[CrossRef][Medline] [Order article via Infotrieve]
31. Bystrom, C. E., Pettigrew, D. W., Branchaud, B. P., O'Brien, P., and Remington, S. J. (1999) Biochemistry 38, 3508-3518[CrossRef][Medline] [Order article via Infotrieve]
32. Orr, G. A., and Knowles, J. R. (1974) Biochem. J. 141, 721-723[Medline] [Order article via Infotrieve]
33. Vas, M., and Batke, J. (1984) Eur. J. Biochem. 139, 115-123[Abstract]
34. Williams, D. M., Jakeman, D. L., Vyle, J. S., Williamson, M. P., and Blackburn, G. M. (1998) Bioorg. Med. Chem. Lett. 8, 2603-2608[CrossRef][Medline] [Order article via Infotrieve]
35. Sherman, M. A., Fairbrother, W. J., and Mas, M. T. (1992) Protein Sci. 1, 1-9[Free Full Text]


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