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 trans- formation 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 mole- cule 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 estab- lished 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).