Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis

Abstract

For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.pericyclic reaction | catalysis | enzyme mechanism | near attack conformation | X-ray crystal structures

Quantum chemical modeling of the reaction path of chorismate mutase based on the experimental substrate/product complex

“…A quantum chemical model of the BsCM active site was prepared based on the substrate complex of the BsCM Arg90Cit variant (PDB‐ID http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3ZP7 ). QM simulations were performed at the DFT (B3LYP) level in analogy to a study on 4‐oxalocrotonate tautomerase .…”

“…All geometries presented in this study were optimized using Gaussian09 at the DFT B3LYP/6‐31G(d) level (see Table S1 and supplementary coordinates). For the models, we selected the residues comprising the active site of BsCM Arg90Cit of the substrate/product‐bound structure (PDB‐ID http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3ZP7 ), in accordance with the methodology described by Sevastik and Himo . nciplot 2.0 was used to visualize the nonbonding interactions of all residues, which allowed removal of noncontributing atoms (e.g.…”

“…The recent high‐resolution crystal structures of wild‐type and Arg90Cit CM provided an ideal basis for improved simulations. This is true in particular for the elusive substrate complex of BsCM, which was recently trapped and refined to a resolution of 1.7 Å .…”

“…The large variation in computational models of the CM Arg90Cit active site [22,27,28,39] suggests that the structural basis for accurate CM simulations has so far been limited. The recent high-resolution crystal structures of wild-type and Arg90Cit CM [40] provided an ideal basis for improved simulations. This is true in particular for the elusive substrate complex of BsCM, which was recently trapped and refined to a resolution of 1.7…”

“…A [40]. Given the choice between different modeling methods, we deliberately chose a QM-only method over QM/MM simulations.…”