Direct Look at the Electric Field in Ketosteroid Isomerase and Its Variants

Abstract

Enzymes catalyze a number of reactions with high efficiency and stereoselectivity. It is thought that strong, direct, and permanent electric fields within the active site of the enzyme contribute to the superb catalytic efficiency of enzymes. This effect is called electrostatic preorganization. Most often, electrostatic preorganization is analyzed by evaluating the local electric field at discrete points, such as a bond center, using, for example, vibrational Stark spectroscopy. However, the protein macromolecule creates a significantly more complicated heterogeneous electric field that affects the entire active site, whose total change density thus gets perturbed, with the implications for the catalytic mechanism. We present a global distribution of streamlines method to analyze the topology of the heterogeneous electric fields in within an enzyme active site. We focus on ketosteroid isomerase (KSI), an enzyme known to produce a field on the order of 100 MV/cm along the critical carbonyl bond in the steroid substrate. We investigate how mutations known to cause activity changes, as well as applied small external electric fields perturb the electric fields in the KSI active site. Where classical single-point analysis failed, using our method allowed us to properly correlate global changes in the electric field to changes in the reaction barrier. We were able to show that topologically similar local electric fields had similar reaction barriers.

Can Electric Fields Drive Chemistry for an Aqueous Microdroplet?

“…In particular optimal alignment of electric elds with large eld strengths can directly affect the energy pro le of chemical reactions through the chemical bonds of the transition states that are well aligned, and thus can signi cantly change the reaction product selectivity and catalysis. 23,26,30,31,32,33,34 When we project the electric elds onto the water molecule chemical bonds as a proxy reaction, we nd that the electric eld alignment with the free O-H bonds of surface molecules increases by ~ 30 MV/cm over that of hydrogen-bonded waters in the inner droplet, with eld strengths which can certainly activate strong bonds or break weak chemical bonds altogether. Our works shows that the nature of chemical reactivity at the air-water interface is not just about structural organization, but about electronic organization at the interface, and supports recent work that has suggested an alternative explanation for surface pH involving charge transfer 35 .…”

Advances in optimizing enzyme electrostatic preorganization

“…In both the KSI and Diels-Alder reactions, we have shown that topologically similar electric fields correspond to similar barriers of the corresponding reaction [19,47]. Furthermore, we showed how fields only at discrete points (which is what is often computed/measured) do not fully capture a relationship to the reaction barrier [47]. Instead, a more global, 3-D geometry of the electric field around relevant bonds are predictive of the reactivity.…”

Catalytic Principles from Natural Enzymes and Translational Design Strategies for Synthetic Catalysts

“…Indeed, both contributions are by definition intimately coupled for determining the reaction rates, as better chemical positioning should correlate with better electric field alignment. 43 , 44 In fact we found that the chemical positioning (i.e., the optimization of the active site base that interacts directly with the substrate) overwhelmingly improved the electric field alignments relative to the original design after 7 rounds of LDE of the catalytic base in KE07-R7. 27 …”