Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis Daniel Burschowskya, André van Eerdea, Mats Ökvista, Alexander Kienhöferb, Peter Kastb,1, Donald Hilvertb,1, and Ute Krengela,1 aDepartment of Chemistry, University of Oslo, NO-0315 Oslo, Norway; and bLaboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland Edited by Arieh Warshel, University of Southern California, Los Angeles, CA, and approved October 30, 2014 (received for review May 8, 2014) For more than half a century, transition state theory has provided ability of a protein to populate this reactive ground state con- a useful framework for understanding the origins of enzyme former has been proposed to account almost entirely for the ef- catalysis. As proposed by Pauling, enzymes accelerate chemical ficiency of CM catalysis (15–17). reactions by binding transition states tighter than substrates, Structural studies have shown that CMs use an extensive thereby lowering the activation energy compared with that of array of hydrogen-bonding and electrostatic interactions to the corresponding uncatalyzed process. This paradigm has been bind the pseudodiaxial substrate conformer (18–20). Sterically challenged for chorismate mutase (CM), a well-characterized constraining chorismate in a “near attack conformation” metabolic enzyme that catalyzes the rearrangement of chorismate (NAC) (15–17, 21), in which the reacting centers are confined to prephenate. Calculations have predicted the decisive factor in to contact distances, would be expected to increase the prob- CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we ability of reaction. Ground state destabilization through con- show, in contrast, that a sluggish variant of Bacillus subtilis CM, formational compression has been shown to afford large rate in which a cationic active-site arginine was replaced by a neutral accelerations for Claisen rearrangements in synthetic model citrulline, is a poor catalyst even though it effectively preorganizes systems (22). Based on molecular dynamics (MD), quantum me- chorismate for the reaction. A series of high-resolution molecular chanics/molecular mechanics (QM/MM), and thermodynamic snapshots of the reaction coordinate, including the apo enzyme, integration studies, Bruice and coworkers estimated that selec- and complexes with substrate, transition state analog and prod- tive NAC formation at the enzyme active site accounts for ∼90% uct, demonstrate that an active site, which is only complementary of the kinetic advantage provided by CM and concluded that in shape to a reactive substrate conformer, is insufficient for ef- transition state binding is unimportant for catalysis in this system fective catalysis. Instead, as with other enzymes, electrostatic sta- (15–17). In contrast, other QM/MM studies (23, 24), including bilization of the CM transition state appears to be crucial for semiempirical and higher level calculations, have suggested that achieving high reaction rates. NAC formation is the result of transition state stabilization, not the source of catalysis. Polar active site residues, most notably a cat- pericyclic reaction | catalysis | enzyme mechanism | near attack conformation | ionic arginine or lysine positioned next to the ether oxygen of the X-ray crystal structures Significance early seven decades ago, Pauling proposed that an enzyme Npreferentially binds and stabilizes the transition state of the chemical reaction it catalyzes relative to the substrate in the Chorismate mutase (CM) is a textbook model for enzyme ca- ground state (1). As a consequence, the activation energy is low- talysis. Although it promotes a simple unimolecular reaction, the origins of its 2-million–fold rate acceleration have been ered for the catalyzed versus the uncatalyzed process, giving rise to debated for decades. The relative importance of electrostatic large rate accelerations. This simple model has successfully in- transition state stabilization versus ground state destabilization formed the development of potent enzyme inhibitors (2), catalytic has been a particularly contentious issue. High-resolution crys- antibodies (3), and computationally designed catalysts (4, 5). tallographic snapshots of an engineered CM variant and its Nevertheless, due to the complexity of even the simplest enzy- complexes with substrate, transition state analog, and product matic systems, a quantitative understanding of the energetics of now provide strong experimental evidence that properly posi- catalysis has remained elusive. The relative contributions of elec- tioned active-site charges are essential in this system and that trostatic, steric, proximity, and solvent effects to transition state preorganization of the substrate in a reactive conformation stabilization and reactant destabilization, as well as the role of contributes relatively little to catalysis. A proper understanding dynamic motions and tunneling, are still heatedly debated (6–10). of the role of electrostatics in this and other enzymes is im- Chorismate mutase (CM) has become a popular model system portant for ongoing efforts to design new enzymes de novo. for studies on enzyme catalysis. It accelerates the unimolecular conversion of chorismate (1) to prephenate (3) (Fig. 1A), a key Author contributions: D.B., P.K., D.H., and U.K. designed research; D.B., M.Ö., and U.K. step in the biosynthesis of tyrosine and phenylalanine, 2 × 106-fold performed research; A.K. contributed new reagents/analytic tools; D.B., A.v.E., M.Ö., and over the spontaneous reaction in water (11). Both the enzymatic U.K. analyzed data; and D.B., A.v.E., M.Ö., A.K., P.K., D.H., and U.K. wrote the paper. and nonenzymatic processes have been shown to be concerted The authors declare no conflict of interest. Claisen rearrangements with asynchronous chairlike transition This article is a PNAS Direct Submission. states (2) (12, 13). Because chorismate exists in an extended Data deposition: The apo BsCM crystal structure and the Arg90Cit BsCM* complexes have 1a been deposited in the RCSB Protein Data Bank, www.rcsb.org (PDB ID codes 3ZOP, 3ZP4, pseudodiequatorial conformation ( ) in aqueous solution (14), 3ZP7, and 3ZO8). accessing the transition state first requires that the substrate un- 1To whom correspondence may be addressed. Email: [email protected], kast@ dergo an energetically unfavorable conformational change to org.chem.ethz.ch, or [email protected]. 1b populate a rare pseudodiaxial conformer ( )inwhichtheenol- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pyruvyl side chain is positioned over the cyclohexadiene ring. The 1073/pnas.1408512111/-/DCSupplemental. 17516–17521 | PNAS | December 9, 2014 | vol. 111 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1408512111 Downloaded by guest on October 1, 2021 of the Arg90Cit variant is not due to inferior transition state -O C A - - - ‡ 2 CO O C O2C 1 2 2 stabilization but rather to major structural rearrangements at the 6 O O 2 O - - - active site that adversely affect the efficiency of forming 5 CO CO CO2 4 2 2 HO 3 O 9 reactive conformers. + - Here we present structural evidence that bolsters the findings CO2 HO HO HO (1a)(1b)(2)(3) from biochemical characterization of the Arg90Cit mutant. High- B C resolution crystallographic snapshots of BsCM and its ligand Arg7 complexes provide direct insight into key species along the re- Tyr108 action coordinate. As seen with many other enzymes (8), elec- HN + NH2 trostatic transition state stabilization rather than reactant HO Xaa90 NH 2 - O destabilization appears to be the determining factor in CM NH O + catalysis. H2N NH2 O - X NH O HN 2 Results O Glu78 Arg63 O- HO (4) To resolve the debate about the relative importance of transition H state stabilization for CM catalysis, we set out to determine the O N Cys75 crystal structure of Arg90Cit BsCM* in its apo form and in O complex with mechanistically relevant ligands. In addition to D Arg90Cit, this variant contains a second substitution (Asp102Glu) that was introduced for semisynthetic production; this change has no affect on activity (25) but serves to distinguish the BsCM* from the WT BsCM samples. Well-diffracting crystals of WT BsCM and Arg90Cit BsCM* (Fig. S1) were obtained under very similar conditions, and the structures of both apo enzymes were solved to a resolution of 1.6 Å. The models include residues 1–117 of the 127-residue enzyme, with residues 2–115 showing well-defined electron density. R/Rfree values are 0.14/0.17 for WT BsCM and 0.17/0.22 for apo Arg90Cit BsCM*. As found previously (18), BsCM adopts a homotrimeric pseudo-α/β-barrel fold with the three active sites positioned at the subunit interfaces (Fig. S2). The overall structures of WT BsCM and Arg90Cit BsCM* are identical within error limits Fig. 1. CM reaction and enzyme active site. (A) The Claisen rearrangement (rms difference of 0.3 Å for Cα of residues 2–115, comparing of chorismate (1a and 1b) to prephenate (3) occurs via a chair-like pericyclic + trimers) and, even in the active site, the two structures super- transition state (2). (B) Schematic view of the active site of BsCM* (X = NH2 ) = impose well. For Arg90Cit BsCM* we note a somewhat greater and Arg90Cit BsCM* (X O) in complex with the conformationally con- conformational freedom for the active-site residues compared strained transition state analog 4.(C)Aσ -weighted 2F − F map of the A o c with WT BsCM, with residues Arg7, Cys75, Glu78, and Cit90 Arg90Cit BsCM* active site in complex with 4. The electron density is con- A toured at 1.5 σ; for clarity, portions of the enzyme obscuring the view of the alternating between two conformations (Fig. 2 ). These con- ligand are not shown. Residues from different subunits are shown in light formational states are largely coupled through an extensive and dark green. Dashed lines indicate H-bonding contacts. A water mole- hydrogen-bonding network, although some variation between cule, bridging the carboxylate groups of the ligand, is shown as a red sphere.