Electrostatic Edge: Decrypting the Near-Perfect Catalytic Efficiency of Fumarase

Fumarase is among the most proficient enzymes and provides a 1015 fold rate enhancement in catalyzing the reversible hydration/dehydration reaction of fumarate/malate. Despite its biological significance, to date, no studies have explained the mechanism and massive catalytic efficiency that lies very close to the diffusion limit. In this report, we present a comprehensive computational study of the iron-independent class II fumarase by employing DFT calculations, MD simulations, QM cluster models, and QM/MM calculations. A carbanionic pathway is found to underlie the catalytic mechanism, both in the aqueous medium and the protein, supported by an extensive hydrogen bond network with the polar substrate at the active site of fumarase. The protein scaffold, beyond the catalytic residues and the active site, is found to have a profound electrostatic effect on amplifying the rate of this reversible reaction. The enormous catalytic efficiency is traced back to a strong electric field at the active site, which has evolved for the selective stabilization of all the higher energy intermediates and transition states along the reaction path compared to the reactant and product. Furthermore, the detrimental effect on catalytic performance upon disruption of the preorganized active site has been investigated through mutational studies. These results underscore the pivotal role of the intrinsic electric field of the enzyme in driving the near-perfect catalytic efficiency of fumarase and provide key insights into enzymatic olefin hydration reactions.