Near Attack Conformers Dominate Β-Phosphoglucomutase Complexes Where Geometry and Charge Distribution Reﬂect Those of Substrate

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Near attack conformers dominate β-phosphoglucomutase complexes where geometry and charge distribution reﬂect those of substrate

Joanna L. Grifﬁna, Matthew W. Bowlerb,1, Nicola J. Baxterc, Katherine N. Leighd, Hugh R. W. Dannatta, Andrea M. Hounslowa, G. Michael Blackburna, Charles Edwin Websterd, Matthew J. Cliffa, and Jonathan P. Walthoa,c,1

aDepartment of Molecular Biology and Biotechnology, Krebs Institute, University of Shefﬁeld, Shefﬁeld S10 2TN, United Kingdom; bStructural Biology Group, European Synchrotron Radiation Facility, F-38043 Grenoble Cedex, France; cManchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7DN, United Kingdom; and dDepartment of Chemistry, University of Memphis, Memphis, TN 38152-3550

Edited by Sun Hur, Harvard Medical School, Boston, MA, and accepted by the Editorial Board March 14, 2012 (received for review October 14, 2011)

Experimental observations of fluoromagnesate and fluoroalumi- (ΔSt+r) and free-energy barriers resulting from steric or electro- nate complexes of β-phosphoglucomutase (β-PGM) have demon- static interactions (ΔGSE). strated the importance of charge balance in transition-state For catalyzed reactions, enzymes reduce the entropic barrier stabilization for phosphoryl transfer enzymes. Here, direct obser- (ΔSt+r) for the reaction (7, 8), and this will reduce ΔGNAC. vations of ground-state analog complexes of β-PGM involving tri- Whether enzymes further stabilize NACs by preferentially re- fluoroberyllate establish that when the geometry and charge ducing ΔGSE, and whether such stabilization has a role in catal- distribution closely match those of the substrate, the distribution ysis, however, remains controversial (9–11). Specifically, the † of conformers in solution and in the crystal predominantly places contention is whether the enzymatic rate enhancement (ΔΔG )is the reacting centers in van der Waals proximity. Importantly, two dominated by enzymatic stabilization of a NAC (ΔΔGNAC)or variants are found, both of which satisfy the criteria for near attack whether ΔΔGNAC is a minor consequence of enzyme stabilization conformers. In one variant, the aspartate general base for the re- of the chemical transition state (ΔΔGchem). NAC stabilization action is remote from the nucleophile. The nucleophile remains can be said to be an important effect if ΔΔGNAC is the principal † BIOCHEMISTRY protonated and forms a nonproductive hydrogen bond to the component of ΔΔG , as suggested for chorismate mutase (where † phosphate surrogate. In the other variant, the general base forms ΔΔG has been calculated to be approximately equal to a hydrogen bond to the nucleophile that is now correctly orien- ΔΔGNAC) (9). However, in a model where stabilization of the tated for the chemical transfer step. By contrast, in the absence of chemical TS is the predominant catalytic effect (i.e., ΔΔGchem >> substrate, the solvent surrounding the phosphate surrogate is ΔΔGNAC), ΔΔGNAC may nonetheless be substantial because the arranged to disfavor nucleophilic attack by water. Taken together, complementarity of the enzyme to the TS will lower the energies the trifluoroberyllate complexes of β-PGM provide a picture of how of species with similar chemistry to the TS (10). A major problem the enzyme is able to organize itself for the chemical step in catal- in addressing this controversy has been the difficulty of obtaining ysis through the population of intermediates that respond to in- experimental evidence for NACs. creasing proximity of the nucleophile. These experimental obser- Enzymes that catalyze reactions that are inherently very slow, vations show how the enzyme is capable of stabilizing the reaction especially because of strong repulsion between substrates, are pathway toward the transition state and also of minimizing arguably the most likely to deploy a high degree of stabilization of unproductive catalysis of aspartyl phosphate hydrolysis. NACs to avoid large barriers to the development of reaction competent states. Archetypal examples of such enzymes operate ground-state analogues | enzyme mechanism on phosphate monoesters, where their extreme kinetic stability to spontaneous hydrolysis leads to calculated lifetimes of up to 1012 y (12). β-Phosphoglucomutase (β-PGM) is a well-studied example he stabilization of transition states (TS) is central to the high − reaction rates achieved by enzymes (1–3). Most studies to date of such an enzyme, where a phosphoryl group (PO3 ) is trans- T β β have focused on TSs that include a chemical transformation step, ferred between -glucose 1,6-bisphosphate ( G1,6P) and an as- where the reacting species have some partial chemical bonding partate (D8) side chain of the enzyme (13, 14). Phosphoisomerism character. However, enzymes also have to avoid substantial bar- is achieved through the release of either glucose 6-phosphate β β riers associated with nonchemical transformation steps and hence (G6P) or -glucose 1-phosphate ( G1P) from the resulting phos- 1 may be required to stabilize other regions of the complex energy phoenzyme complex, according to Scheme . Work to date has β surface corresponding to the reaction coordinate. The concept of focused on probing the reaction mechanism of -PGM through the near attack conformers (NACs) was introduced as a useful tool to help analyze these different roles, by partitioning chemical transformation and nonchemical transformation steps (4). NACs were Author contributions: J.L.G., M.W.B., N.J.B., K.N.L., G.M.B., C.E.W., M.J.C., and J.P.W. de- fi signed research; J.L.G., M.W.B., N.J.B., K.N.L., H.R.W.D., A.M.H., C.E.W., and M.J.C. per- de ned as species where the reactants have an appropriate ge- formed research; H.R.W.D., A.M.H., and M.J.C. contributed new reagents/analytic tools; ometry for the development of the chemical TS (5, 6). Further- J.L.G., M.W.B., N.J.B., K.N.L., C.E.W., M.J.C., and J.P.W. analyzed data; and M.W.B., N.J.B., more, the component of the TS barrier ascribed to the chemical TS G.M.B., M.J.C., and J.P.W. wrote the paper. was proposed to be relatively constant within systems carrying out The authors declare no conflict of interest. similar chemistry, on the basis of a linear relationship between the This article is a PNAS Direct Submission. S.H. is a guest editor invited by the Editorial (calculated) population of NACs and the corresponding reaction Board. rates (5). Hence, in free-energy terms, the NAC model apportions Data deposition: The atomic coordinates, structure factors, and NMR chemical shifts re- † the TS barrier (ΔG ) between a variable conformational compo- ported in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID Δ codes 2wf8, 2wf9, and 2wfa), and the BioMagResBank, www.bmrb.wisc.edu (BMRB ID nent ( GNAC) and an invariant chemical (covalent) component codes 17851 and 17852). Δ ( Gchem; Fig. 1). For some uncatalyzed reactions, it was proposed 1To whom correspondence may be addressed. E-mail: j.waltho@sheffield.ac.uk or that a major component of the TS free-energy barrier is the pop- [email protected]. ulation of NACs, the instability of NACs in solution being ascribed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. to a combination of rotational and translational entropy barriers 1073/pnas.1116855109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1116855109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 geometry (Fig. S1). The cap and core domains of the enzyme are arranged in an open conformation (Fig. 2A) similar to the crystal structure of apo-PGM (20) (heavy atom rmsd 0.57 Å). The groups − that coordinate the BeF3 moiety are all contributed from the core domain and are the same as the cognate groups in the fully closed PGM-MgF3-G6P TSA complex (20). An additional water molecule coordinates FA (Fig. 3A). The catalytic base, D10, does not − coordinate water molecules in a position to attack the BeF3 moiety, but instead hydrogen bonds with a water bound to the catalytic Mg2+ ion and with the side chain Oγ and backbone amide of T16, as in other structures of β-PGM in the absence of sugars Fig. 1. Free-energy diagram depicting the NAC model for transition-state [note that a previously proposed aspartyl phosphate structure (28) − barriers. The TS is proposed to be comprised of a conformational component is most likely a PGM-AlF4 complex (20)] (28, 29). (ΔG ) and the remaining chemical component involving breaking and NAC The solution behavior of the PGM-BeF3 GSA complex is making of bonds (ΔG ). The effect of enzymes is to reduce the TS barrier chem distinct from either apo-PGM or the PGM-MgF3-G6P TSA height (blue line) through stabilization of NACs and/or reducing the chem- complex (16). The backbone NMR resonances of 208 residues ical barrier by specifically binding the TS. were assigned (SI Text), which included many residues in active site loops and in the hinge regions (in particular, D8-A17, L44- apo introduction of metal fluoride transition-state analogs (TSAs) and L53, and S114-N118) that were not assignable for -PGM computational analysis based on the derived structural data (15– owing to millisecond dynamics (16). Indeed, the resonances − unassigned in apo-PGM show the largest chemical shift changes 21). In particular, trifluoromagnesate (MgF3 ) has been established as a very close steric and electronic mimic of the planar between the PGM-BeF3 GSA and the PGM-MgF3-G6P TSA − complexes (particularly the hinge residues D15–A17), indicating PO3 group (16, 20, 22–24) that represents the energy maximum as − − that the binding of the BeF moiety substantially reduces con- phosphorus moves through the plane of the three PO oxygen 3 3 formational dynamics associated with interdomain movement. atoms (15, 21). In addition to providing atomic resolution struc- Overall, the chemical shift changes between the PGM-BeF GSA tures of β-PGM in a near TS conformation, the introduction of 3 and the PGM-MgF -G6P TSA complexes show an almost per- metal fluorides allows 19F NMR to provide measures of the 3 fectly linear relationship (R = 0.94; Fig. S2) with those between electronic and protonic environment at the catalytic center (20). apo-PGM and the PGM-MgF -G6P TSA complex. The slope of Examination of stable ground-state analog (GSA) complexes 3 0.94 indicates that the PGM-BeF3 GSA complex lies close to the has the potential to determine whether phosphoryl transfer dominant conformation of the apo protein, and hence is in enzymes do impart a high degree of stabilization of NACs, and agreement with the conformation in the crystal. This agreement comparisons with studies of TSA complexes should allow an ex- is further supported by backbone dihedral angle predictions ploration of the relationship between NAC and TS stabilization. based on chemical shifts, using TALOS (30). fl − Tri uoroberyllates (BeF3 ) have been established as GSAs for The 19F NMR spectrum of this complex shows three resolved phosphate groups in a number of proteins (25–27). In these GSA − peaks for the protein-bound BeF3 moiety (FA: −150.2, FB: complexes, the Be atom bonds to the bridge O atom in place of −151.5, and FC: −178.7 ppm; Fig. 4A, Fig. S3, and Table S2). The the P atom, and three F atoms substitute three nonbridge O resonances were assigned using {19F} 1H NOE experiments (Fig. − – atoms. The typical inorganic Be Fbond(1.5 1.6 Å) is of similar S4) and deuterium solvent-induced isotope shifts (SIISs; Table length to typical P–O bonds in phosphates, producing surrogates S2). Though the three resonances are well resolved, the {19F} 1H with comparable geometry, near-obligate tetrahedral organiza- NOE experiments indicate exchange between the 19F resonances tion, and the same net charge (26). Here, by combining high- on a subsecond timescale (SI Text), corresponding to slow rota- − resolution X-ray crystal structures and NMR data, we report that tion of the BeF moiety. The chemical shift of F is significantly − β 3 C BeF3 complexes of -PGM with glucose phosphate deploy a high upfield of the other resonances and has a very low SIIS (Table degree of stabilization of NACs. Indeed, multiple conformations − S2), indicating that it has relatively high electron density and low containing BeF3 are populated, with different juxtapositions of proton density (18, 20). These properties are reminiscent of the the reacting moieties demonstrating the importance in enzyme FC resonance in the PGM-MgF3-2-deoxyG6P TSA complex (18), catalysis of the stabilization of intermediates throughout the where it is not hydrogen bonded to an OH group and is co- reaction. ordinated only by the catalytic Mg2+. An equivalent arrangement is present in the crystal of the PGM-BeF3 GSA complex where, Results surprisingly, the positioning of water molecules in the active site Phosphoenzyme Analog. An analog of the phosphoenzyme in- area denies solvation of the FC atom (Fig. 3A). termediate (termed the PGM-BeF3 GSA complex) is formed on 2+ − addition of Be and F to apo-PGM. This complex crystallized in Near Attack Conformer I. The addition of G6P to the PGM-BeF3 the orthorhombic space group P212121 similar to the native struc- GSA complex to form the PGM-BeF3-G6P GSA complex results ture (20), and the structure was solved by molecular replacement to in substantial changes in the protein conformation. The backbone − 1.65 Å resolution (Table S1). When unrestrained, the BeF3 resonances of 200 residues were assigned; the missing resonances moiety refined to an average Be–F bond length of 1.56 Å (this is were principally in the cap domain and the interdomain hinges. significantly shorter than the commonly used value of 1.76 Å); it is The protein conformation is distinct from complexes studied jΔδ j coordinated by the reactive aspartate, D8, and has tetrahedral previously. The amide chemical shift changes that are large ( N > 0.3 ppm) between the PGM-BeF3 GSA and the PGM-MgF3- G6P TSA complexes correlate with the changes between the PGM-BeF3 GSA and the PGM-BeF3-G6P GSA complexes with a coefficient of 0.76 (Fig. S2) and a slope of 0.56 ± 0.04. For example, the largest change in ΔδN between the PGM-BeF3 GSA complex and the PGM-BeF3-G6P GSA or PGM-MgF3-G6P TSA complexes is that of a critical hinge residue, D15, which moves by Scheme 1. Reaction scheme of β-PGM. 4.6 ppm and 7.5 ppm, respectively; this indicates that the PGM-

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1116855109 Grifﬁnetal. Downloaded by guest on September 25, 2021 Fig. 2. Changes in domain closure between the triﬂuoroberyllate complexes of β-PGM. The overall architecture of β-PGM is shown as a ribbon, with the core

(red) and cap (green) domains indicated: (A) the PGM-BeF3,(B) the PGM-BeF3-G6P NAC I, and (C) the PGM-BeF3-G6P/G1P NAC II crystal structures. Shown in all panels in white ribbons are the apo-PGM and PGM-MgF3-G6P TSA structures (20) overlaid on the core domain, to show the extent of domain closure associated with the new structures.

BeF3-G6P complex shows ∼50–60% of the full domain closure. TSA complex. This conformation is thus termed NAC I. In- However, there are exceptions to this correlation (Fig. S4). For triguingly, both α-andβ-anomeric forms of G6P are populated in example, residues closely associated with metal fluoride binding, NAC I (50% α, 50% β; Fig. S1), i.e., the mechanism of anomer such as D10, G46 and A113, show distinct chemical shift changes selection present in the PGM-MgF3-G6P TSA complex is not between each of the three complexes. active in NAC I. In the absence of the catalytic base, the phosphate The PGM-BeF3-G6P GSA complex crystallized in the ortho- surrogate acts as a hydrogen bond acceptor for the 1-OH of G6P, P rhombic space group 212121, with unit cell dimensions that are with different F atoms accepting the hydrogen bond for each of the different from previously solved structures, and the structure anomers bound. solved to 1.4 Å by molecular replacement (Table S1). When un- The 19F NMR spectrum of the PGM-BeF -G6P GSA com- − fi 3 restrained, the BeF3 moiety re ned with an average bond length fl

plex re ects the dominance of NAC I within the solution en- BIOCHEMISTRY of 1.55 Å. The cap and core domains are marginally more closed 15 semble. Resonances corresponding to complexes containing than estimated from N solution NMR measurements, having either α-orβ-anomer of G6P are present (F : −150.2 ppm; 75% of the 33° domain rotation between the PGM-BeF GSA and left 3 F : −153.6 ppm; Fig. 4B, Fig. S3,andTable S2). The in- the PGM-MgF -G6P TSA complexes (Fig. 2B). However, the right 3 dividual 19F resonances are not resolved within each complex; backbone dihedral angles in the crystal closely match predictions 13 rather, the interconversion rate of fluorides in either complex based on C NMR chemical shifts, using TALOS (30). The cat- fi alytic base, D10, is still not engaged with the nucleophile for the is suf ciently fast to cause line broadening and convergence of B chemical shifts (millisecond timescale rotation). The increased phosphoryl transfer reaction (the 1-OH of G6P; Fig. 3 ); it − maintains hydrogen bonds with a water molecule bound to the rotational rate of the BeF3 moiety compared with the PGM- fi 19 1 catalytic Mg2+ ion, and with the backbone amide of T16, and also BeF3 GSA complex is con rmed in { F} H NOE experiments, hydrogen bonds with the 2-OH of G6P. Significantly, the distance where both Fleft and Fright have NOEs with all of the co- − and inline angle between the nucleophile and the BeF3 moiety ordinating hydrogen atoms in NAC I (Fig. S4). Based on the are well within the criteria for a NAC (31) (Table S3). For ex- population of the two sugar phosphates observed in the crystal ample, the oxygen donor (D8:Oδ) to acceptor (1-OH) distance structure and the different deuterium SIIS for Fleft and Fright (4.9 Å) indicates a close approach of the 1-OH to the phosphate (Table S2), the resonances are tentatively assigned to the αG6P surrogate, and is comparable with 4.3 Å in the PGM-MgF3-G6P and βG6P complexes, respectively.

Fig. 3. The active site of PGM in the various complexes. Selected active-site residues in the crystal structures of (A) the PGM-BeF3,(B) PGM-BeF3-G6P NAC I, (C) PGM-BeF3-βG1P NAC II, and (D) PGM-BeF3-G6P NAC II GSA complexes are shown as sticks, with polar atoms shown in the standard CPK colors, and water oxygen (red) and Mg (green) atoms shown as spheres. Blue dashes indicate probable hydrogen bonds (distance <3.2 Å), and in the PGM-BeF3-G6P NAC II complex, the nucleophile-Be vector. The residues were identiﬁed as critical to catalysis in Dai et al. (29).

Griffinetal. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 Fig. 4C and Table S2). (The rotational rate of the fluoroberyllate moiety again is slowed such that individual resonances are resolved.) The residual activity of β-PGM in the presence of excess fluoroberyllate indicates the reversibility of the fluoroberyllation of the nucleophilic aspartate D8. The effects of the different glucose phosphates on the protein conformation were examined using 1H,15N TROSY spectra. Importantly, key active site residues, such as the general base, D10, and the important hinge residue, D15, are substantially affected by the switch between G6P and β-G1P binding, in line with the transition between NAC I and NAC II observed in the crystal structures.

Influence of Fluoroberyllate. To establish the extent to which the nature of the complexes may be influenced by the use of a phosphate surrogate rather than the true reaction intermediate, atomic charges were calculated for the two moieties, 19 Fig. 4. F NMR spectra of the GSA complexes. (A) PGM-BeF3 GSA complex, both as isolated entities and in the context of the protein. A (B) PGM-BeF3-G6P complex, and (C) PGM-BeF3-G1P complex. The PGM-BeF3 strategy that involved correlating Mulliken charges and atomic GSA complex contained 0.5 mM β-PGM, 3 mM BeCl2, and 10 mM NH4F. The polar tensor-based charges was used (SI Text, Fig. S6). In the PGM-BeF3-G6P and PGM-BeF3-G1P GSA complexes were formed by the ad- active site, the fluoride ions (average charge = −0.67) carry dition of 10 mM G6P and 10 mM β-G1P, respectively. Asterisked resonances ∼70% of the negative charge of the corresponding oxygen atoms arise from inorganic fluoride species. Protein-bound resonances are labeled (average charge = −0.95), and the beryllium atom (charge = – B FA FC by analogy with the PGM-MgF3-G6P TSA complex (20), and in labeled +1.2) carries ∼60% of the positive charge of the corresponding F and F , owing to rotational averaging. left right phosphorus atom (charge = +2.1; Table S4). Hence, though the fluoroberyllate moiety carries approximately the same overall charge as the genuine reacting species, it is somewhat Near Attack Conformer II. The PGM-BeF3-G6P GSA complex also crystallized in a second crystal form, with unit cell dimensions scaled down in terms of its internal charge separation. much closer to crystals of the PGM-MgF -G6P TSA complex 3 Discussion (Table S1). The structure was solved to 1.2 Å by molecular re- − fl placement. When unrestrained, the BeF moiety refined with The use of uoroberyllate as a GSA for phosphoryl transfer has 3 β an average bond length of 1.56 Å. The protein conformation allowed the characterization of states of -PGM that have not been accessed by other means. The instability of aspartyl phos- (Fig. 2C) was found to be very similar to the PGM-MgF3-G6P TSA complex structure with 0.6 Å heavy-atom rmsd, and a 33° phates in solution (32) severely hampers their observation by spectroscopic and crystallographic methods. However, the near- domain reorientation relative to apo-PGM. The electron density − for the sugar phosphate (Fig. S1) revealed one β-G6P and two obligate tetrahedral coordination of the BeF3 moiety and its identical net charge with that of the corresponding phosphoryl β-G1P complexes in a 2:1:1 ratio (β-G6P dominating). The − group makes it a close surrogate (26). The BeF moiety readily β-G6P moiety is positioned such that the oxygen acceptor (1- 3 assembles in the active site of β-PGM without the requirement OH) is 4.4 Å from the donor (D8:Oδ), which is comparable with for chemical synthesis of an analog, and the accessibility of the separation in the PGM-MgF -G6P TSA complex (4.3 Å). 3 multiple GSA complexes, in combination with the unliganded The β-G6P complex again fulfills the criteria for a NAC (Table enzyme and the TSA complex, allows stationary states along the S3) and is thus termed NAC II (Fig. 3D). The general base, D10, reaction coordinate of β-PGM catalyzed phosphorylation of has the same conformation as in the PGM-MgF3-G6P TSA glucose phosphate to be examined. complex, where it forms a hydrogen bond with the nucleophile, The phosphoenzyme in the absence of glucose phosphate is with T16:Oγ, and with a water molecule that is also coordinated A A η ɛ γ mimicked by the PGM-BeF3 GSA complex (Figs. 2 ,3 , and by Y80:O , H20:N , and S116:O . The aromatic side chains of 4A). In structural terms, the open conformation of the GSA H20 and Y80 stack against each other, whereas H20 packs complex is suitable for access of the substrate to the active site, against the glucose ring and, together with the movement of the whereas the general base that promotes reactivity (the D10 side cap domain between NAC I and NAC II, they reposition the chain) is orientated away from its position in the TSA complex. β β glucose moiety. In contrast to -G6P, -G1P is bound in two Such disruption of general base catalysis should reduce the rate orientations: in one the 6-OH (Fig. 3C), and in the other the 3- − of hydrolysis of the phosphoenzyme resulting from the in- OH, are positioned close to the BeF3 moiety. The donor-ac- advertent activation of water molecules (19, 20, 28, 29). Indeed, β ceptor distances for both -G1P complexes are much longer (5.1 the high resolution of the PGM-BeF GSA complex structure β 3 and 5.6 Å) than in the -G6P complex and the 3- or 6-OH hy- (1.65 Å) allows the arrangement of water molecules in the active β drogen bonds to FC. Therefore, only -G6P has the nucleophilic site to be determined accurately. In the crystal, no solvent oxygen OH positioned for inline attack on the beryllium surrogate for atoms are closer than 3.4 Å to the Be atom within the aspartyl the phosphorus center of the aspartyl phosphate (Table S3). phosphate surrogate. In solution, the dehydration of FC in this The production of a β-G1P complex in the NAC II crystal complex, as indicated by its NMR properties, further establishes structure indicates that significant mutase activity persists in the that water molecules are significantly restricted in their ability to 2+ − presence of Be and F , because no β-G1P had been added to form hydrogen bonds with potential donors or acceptors in the the crystallization solution for the PGM-BeF3-G6P GSA com- active site. Moreover, the reduction in conformational exchange fi 31 plex. Mutase activity was con rmed in solution using P-NMR in the PGM-BeF3 GSA complex (relative to all of the other (Fig. S5), which also demonstrated that β-G1P binds in prefer- β-PGM complexes studied) is consistent with precise control of ence to β-G6P even when G6P is in excess. Indeed, the distri- solvation. However, the protection of phospho-β-PGM is not bution of PGM-BeF3-glucose phosphate complexes changes on absolute, and kinetic analysis (19) reveals that its aspartyl a timescale of hours. Starting with G6P, the 19F NMR spectrum phosphate has a hydrolysis half-life of ∼30 s. B (Fig. 4 ) slowly transforms into the spectrum corresponding to Initial binding of G6P to the PGM-BeF3 GSA complex causes the β-G1P GSA complex (δF −147.7, −148.8, and −165.1 ppm; a substantial perturbation of the phosphate surrogate, but the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1116855109 Griffinetal. Downloaded by guest on September 25, 2021 general base for the reaction, D10, remains remote from the in the transition between the conformations denoted NAC I and nucleophile (the 1-OH of G6P; 4.4 Å; Figs. 2B,3B, and 4B). The NAC II. These experimental observations marry well with quan- interaction of β-PGM with the sugar phosphate induces sub- tum mechanics/molecular mechanics predictions of the chemical – stantial domain closure, with residues L44 R49 and S52 from the barrier in this enzyme, built on the basis of the PGM-MgF3-G6P cap domain and S116 and K117 from the core domain having TSA complex structure (15, 21). In both computational studies, it interactions with the sugar in the PGM-BeF3-G6P GSA complex was calculated that the 1-OH nucleophile of β-G6P remains closely similar to those in the PGM-MgF3-G6P TSA complex. protonated except when the phosphorus atom is located signifi- Though the relative positions of the nucleophile and the phos- cantly on the sugar side of the medial plane of the three phosphate surrogate equate to a NAC, the chemistry does not. The phoryl oxygen atoms. remoteness of D10 means that the G6P 1-OH nucleophile The complexes of β-PGM discussed here are based on metal remains protonated, which in turn allows this group to hydrogen fluoride analogs rather than phosphates, and that fact precludes bond to the fluoride surrogates of the phosphate oxygen atoms the direct use of arguments based on precise stability measure- and achieve reduced electrostatic repulsion. The incompleteness ments to resolve unambiguously the controversy over whether of the complex in terms of preparing the chemistry for phos- NACs are the primary targets for stabilization by enzymes or phoryl group transfer is also evident in the accommodation of whether their population is more a side product of TS stabili- both the α- and β-anomers of G6P with similar affinity. This zation. However, the observation that NACs dominate the con- structure most likely represents initial stabilization of NAC be- formational ensemble in β-PGM, when geometry and charge fore catalysis can occur and is the first step in positioning the closely match those of the substrate, points toward a very strong substrates for nucleophilic attack. preference for the enzyme to organize the relevant moieties in The second NAC populated by the PGM-BeF3-G6P GSA readiness for the chemical step. Indeed, the increased calculated complex (NAC II; Figs. 2C and 3D) is correctly orientated for the charge separation in the phosphate moiety compared with the chemical transfer step to β-G6P. In this complex, one of the Oδ fluoroberyllate should scale up the interactions responsible for atoms of D10 forms a hydrogen bond to the nucleophilic 1-OH the overall free-energy change in favor of NAC formation. group and so is positioned to accept its proton. The interaction Though this supports a major contribution to catalysis coming between the D10 side chain and T16:N present in NAC I is broken from enzymatic stabilization of the NAC, the charge dis- as the domains move and the preceding carbonyl O atom (D15:O) tributions in the PGM-BeF -G6P GSA and the PGM-MgF - – 3 3 joins the hydrogen bonding of helix 1 (D15 I31). Indeed, com- G6P TSA complexes are closely similar; the primary difference plete closure of the domains appears to be dependent on the being the relative inaccessibility of d-orbitals in Be and thus its BIOCHEMISTRY disruption of this interaction between D10 and the N terminus of resistance to forming a pentacoordinate, trigonal bipyramid. helix 1, and is key to the general base engaging with the nucleo- Consequently, the beneficial effects of the enzyme on ΔG phile. Between NAC I and NAC II, the sugar ring and the side NAC and ΔGchem are far from independent. Although the true sub- chains of H20 and Y80 move in concert as the domains close, strate complexes are not experimentally accessible, the metal which displaces the 1-OH group such that it is better aligned to fluoride structures provide a platform for in-depth computa- attack the phosphate moiety. Thus, the hydrogen bonds from the − tional assessment of the relative contributions of the enzyme to 1-OH group to the BeF3 moiety and from the 2-OH group to the ΔG and ΔG . Moreover, our experimental results dem- side chain of D10 are swapped to produce hydrogen bonds from NAC chem − onstrate that the enzyme overrides the anion-anion repulsion the 2-OH group to FC of the BeF3 moiety and from the 1-OH fl B D between the uoroberyllate moiety and the oxygen nucleophile, group to the side chain of D10 (Fig. 3 and ). As a consequence, plus its attendant protein side chain (D10), to such an extent that the donor-acceptor distance is reduced from 4.9 to 4.4 Å, which is an almost fully closed conformation (NAC II) is readily popu- within the distance required for a concerted (with respect to lated. This behavior supports the hypothesis that charge balance distance) reaction (33). It is likely that this crystal form is selected between the enzyme active site and the substrate is central to owing to the production of β-G1P via residual mutase activity. The accelerated progression along the reaction coordinate (17, 24). 6-OH proton of β-G1P in NAC II retains the ability to hydrogen Finally, this work establishes that, for phosphoryl transfer, the bond with a surrogate of the phosphate oxygen atoms, similarly to route leading to the formation of a productive, on-enzyme NAC the 1-OH of G6P in NAC I. However, the corresponding domain can have other minima (e.g., NAC I) that reduce repulsion be- arrangement determines that β-G6P is accommodated with better tween reacting groups, which might otherwise result in obligatory alignment to attack the Be atom. Hence, though the two sugars high-energy species on the reaction pathway. appear to prefer different interdomain angles, β-G6P is readily accommodated in the more closed conformation and thus the free Methods energy of this state cannot be substantially greater than for G6P in Lactococcus lactis β fi the NAC I conformation. -PGM was expressed recombinantly and puri ed as de- scribed previously (16). All experiments were recorded in 50 mM K+ Hepes 2 Conclusions buffer (pH 7.2) containing 5 mM MgCl2, 2 mM NaN3, and 10% (vol/vol) H2O at 298 K, unless otherwise stated. Taken together, the trifluoroberyllate complexes of β-PGM provide a picture of how the enzyme is able to organize itself for the NMR. 19F and 1H,15N TROSY experiments (34) were recorded on Bruker chemical step in catalysis through the population of low free-en- Avance 500-MHz and 600-MHz spectrometers, respectively. For assignment, ergy intermediates with increasing proximity of the nucleophile the standard suite of 3D TROSY-based triple-resonance experiments (16) was and, at the same time, minimize inadvertent and deleterious recorded on a Bruker Avance 800-MHz spectrometer. catalysis of aspartyl phosphate hydrolysis. Though the tri- − fluoroberyllate complexes give access to pretransition state Crystallization. β-PGM was prepared at a concentration of 15 mg·mL 1 in 50 + complexes only on one side of the TS—where the enzyme is ac- mM K Hepes (pH 7.2), 5 mM MgCl2, 1 mM azide, 0.1 mM DTT, 10 mM NH4F, and 10 mM BeCl2. For crystallization, the solution was mixed 1:1 with the celerating the cleavage of a phosphate carboxylate anhydride that – is substantially more facile than cleavage of a phosphate mono- precipitant [26 30% (wt/vol) PEG 4000, 200 mM Na acetate, and 100 mM Tris — (pH 7.5)] and placed in sitting-drop crystallization plates. For the PGM-BeF3- ester they show that NAC species are strongly stabilized by the G6P GSA complex, this solution was supplemented with 5 mM G6P (final enzyme. The structures clearly demonstrate the value of main- concentration) before mixing with precipitant. Diffraction data were col- taining protonation of the nucleophile as it moves into proximity lected from cryocooled crystals to between 1.65 and 1.2 Å resolution on an with the phosphate moiety. Production of the chemically com- ADSC Q4R CCD detector on beamline ID14-2 (λ= 0.933 Å) at the European petent state results from the switching of hydrogen bond partners Synchrotron Radiation Facility.

Grifﬁnetal. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. We thank the University of Memphis High Perfor- Biotechnology and Biological Sciences Research Council, the Wellcome Trust, mance Computing Facility and Computational Research on Materials In- National Science Foundation Early Career Development (CAREER) Grant CHE stitute (CROMIUM) for computing support. This work was supported by the 0955723, and the University of Memphis.

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