STRUCTURE NOTE

Crystal Structure of Phosphoglucose Isomerase From PigMuscle and Its Complex With 5-PhosphoarabinonateChristopher Davies1* and Hilary Muirhead2

1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina2Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, United Kingdom

Introduction. Phosphoglucose isomerase (PGI) (EC5.3.1.9) is a key enzyme in glycolysis and gluconeogenesisthat catalyzes the interconversion of glucose 6-phosphate(G6P) and fructose 6-phosphate (F6P) by transfer of acarbon-bound hydrogen between C1 and C2. The mecha-nism is believed to be a proton transfer via a cis-enediolintermediate. Crystallographic structures of the enzymefrom rabbit 1 (Davies and Muirhead, unpublished) andhuman2 suggest that Glu-357 is the base responsible forthis transfer. Other aspects of the reaction mechanism,however, remain sketchy, including the identity of thegroup responsible for catalyzing ring opening.

Interest in PGI has increased in recent years after anumber of discoveries linking it to various cytokine factors[e.g., autocrine motility factor (AMF)].3 However, severalinconsistencies remain; the most notable is how the estab-lished dimeric structure of the enzyme is related to itscytokine form, which appears to be monomeric.

Here we present the structure of PGI from pig musclesolved at 2.5 Å resolution together with a 3.5 Å structure ofthe enzyme bound to the transition-state mimic, 5-phos-phoarabinonate (PAB). A comparison of these structuresreveals conformational changes in the enzyme that occurduring catalysis.

Materials and Methods. PGI from pig muscle crystal-lizes from ammonium sulfate in space group P43212 withone monomer in the asymmetric unit.4 The native dataused in this study were collected at the SRS, Daresbury,using film methods. Data were collected at room tempera-ture from a total of seven crystals and were processed withMOSFLM (CCP4). These data are 75.7% complete to 2.18Å (Table I). Because most of the absent data are at thehigh-resolution end, only data to 2.5 Å were used forrefinement. The structure of pig PGI was solved originallyat 3.5 Å resolution,5 but refinement at higher resolutionwas impeded by a lack of quality phases. With a high-resolution structure of the rabbit enzyme in hand (Daviesand Muirhead, unpublished), a new pig structure wasgenerated by applying a transformation to one monomer ofthe dimeric rabbit structure. The sequence was thenchanged to correspond to that of pig, and the structure was

refined at 2.5 Å resolution, initially using XPLOR but laterwith REFMAC. The final model is composed of 554 resi-dues, 141 water molecules, and 1 sulfate molecule (PDBcode 1GZD). Residues 555–557 are not visible in theelectron density map and are excluded from the model.The crystallographic R and free R factors are 17.5% and24.0%, respectively, with good geometry (Table I). 88.8% ofthe residues lie in the most favored region of the Ramachan-dran plot with no outliers.

Data were collected previously from crystals of pig PGIthat had been crystallized in the presence of PAB6 extend-ing to 3.5 Å resolution. To model the inhibitor-boundenzyme, the pig structure (without the water and thesulfate molecules) was refined using XPLOR and REFMAC.Manual adjustments were made to the model by using theO program. The final model, which contains residues1–555 and one molecule of PAB, has a crystallographic Rfactor of 19.3% (Rfree � 24.9%) with good geometry (PDBcode 1GZV)(Table I). 88.1% of the residues lie in the mostfavored region of the Ramachandran plot with no outliers.

Results and Discussion. Structural studies of PGI frompig muscle first began 30 years ago, culminating in apolyalanine structure at 3.5 Å resolution.5 When thesequence for PGI became available, refinement of thisstructure at higher resolution stalled, largely because ofthe lack of quality phases. The problem was resolved bydetermining a whole new structure, that of rabbit PGI, byheavy atom methods (Davies and Muirhead, unpublished)and using this to correct model errors in the pig structure.Because the original 3.5 Å structure was solved in advanceof any sequence information for the enzyme,5 this modelwas incomplete and contained several connectivity errors.These were corrected in the current structure by reversingthe directions of five �-helices and one �-strand: A9, A14,A15, A16, A17, and B6 (1977 nomenclature). In doing so,

*Correspondence to: Christopher Davies, Department of Biochemis-try and Molecular Biology, Medical University of South Carolina, 173Ashley Avenue, Charleston, SC 29425. E-mail: davies@musc.edu

Received 31 May 2002; Accepted 22 July 2002

Published online 00 Month 2002 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/prot.10255

PROTEINS: Structure, Function, and Genetics 50:577–579 (2002)

© 2002 WILEY-LISS, INC.

the unusual left-handed connection between B4 and B5 nolonger exists. Several new elements of secondary structurewere identified, including an additional �-strand and�-helix in the small domain, which accounts for most of the43 residues missing in the 3.5 Å model.

Pig PGI crystallizes with one monomer in the asymmet-ric unit, whereas the native form of the enzyme is dimeric.Applying a symmetry operation can generate the dimer,and this is prerequisite for viewing either active-siteregion, each of which is comprised of chains from bothmonomers. This arrangement agrees with biochemicaldata showing that the dimer is required for enzymaticactivity7 but is at odds with data suggesting that AMFexhibits PGI enzyme activity and yet appears to exist as amonomer.3

As expected, the structure of porcine PGI is highlysimilar to those of PGIs from other mammalian sources. Itis most highly similar to that of human PGI2: the rootmean square in all main-chain atoms between thesestructures is 0.4 Å. A molecule of sulfate from the crystalli-zation solution is observed in the active site of pig PGI,which appears to mimic the phosphate moiety of theglucose 6-phosphate or fructose 6-phosphate substrates. Asimilar sulfate was also observed in the structure ofhuman PGI,2 and when this structure was compared withthat of native rabbit PGI, some structural differences werenoted in the small domain that were attributed to confor-mational changes induced by sulfate binding. It wassuggested that similar changes would occur when theenzyme recognizes the phosphate moiety of the substrate.The fact that pig PGI also contains bound sulfate and alsoadopts the same conformation in this region supports thishypothesis.

The new structure for pig PGI was used to reexamine 3.5Å data collected from crystals soaked in 5-phosphoarabi-nonate (PAB).6 This is a competitive inhibitor of PGI (Ki �2.7 � 10�7) that mimics the cis-enediolate intermediate.At that time, difference Fouriers established the locationof the active site, but without a complete model, little couldbe inferred about the identity of active-site residues. In thenew structure, the enzyme-inhibitor contacts can be seen

clearly including the putative base catalyst, Glu-357,which contacts one of the oxygens of the C1 carboxylategroup. Comparison of the native and PAB-bound struc-tures shows that residues 512–520 (inclusive), whichcomprise the N-terminal half of helix �23, shift signifi-cantly toward the active site in the inhibitor-bound struc-ture (Fig. 1). A similar change has also been observed inthe crystal structures of rabbit PGI,8 but this is the firsttime a comparison has been made with a native structureof mammalian PGI. Of particular note are the large shiftsof Glu-515 and Lys-518. The new position for Glu-515equates to a 7 Å movement of its epsilon oxygens towardthe active site. Similarly, the zeta nitrogen of Lys-518 hasmoved 3.1 Å to make contact with both O4 and O5 of theinhibitor. Lys-518 likely plays a significant role in thecatalytic mechanism, either by protonating O5 during ringopening or by transferring a proton between the C1 and C2hydroxyls.1

Conclusions. Taken together with previous investiga-tions, these data suggest there are two conformationalchanges associated with the catalytic activity of PGI.Recognition of the phosphate group of the substrate in-duces two loops in the small domain to close down onto theactive site and, near the C-terminus, �23 also movestoward the active site to bring Lys-518 into contact withthe substrate.

TABLE I. X-ray diffraction data and modelrefinement statistics

Native PAB

Resolution range (Å) 31–2.19 17.7–3.5Rmerge (%) 7.3 4.5Completeness (%) 75.7 92.8Resolution range for refinement (A) 15.0–2.5 15.0–3.5% reflections used for Free R set 10.0 5.0No. of protein atoms 4422 4422No. of hetero atoms 5 15No. of waters 141 0R factor (R work � R free)(%) 17.5 19.3R free (%) 24.1 25.0Overall Biso (Å2) 17.2 42.1R.M.S deviation for bond length (Å) 0.018 0.019R.M.S deviation for angles (Å) 1.81 1.98

Fig. 1. The movement of helix �23 on binding the competitiveinhibitor, 5-phosphoarabinonate (PAB). Shown is a backbone superimpo-sition of the native (dark gray) and PAB-bound (light gray) PGI structuresin the active-site region. Important active-site residues are shown as solidbonds for the native structure and dashed bonds for the PAB-boundstructure. The PAB inhibitor is shown in ball-and-stick form. For clarity,most loops in the active site have been removed.

578 C. DAVIES AND H. MUIRHEAD

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CRYSTAL STRUCTURE OF PIG PHOSPHOGLUCOSE ISOMERASE 579