Ketosteroid isomerase provides further support for the idea that enzymes work by electrostatic preorganization

Abstract

One of the best systems for exploring the origin of enzyme catalysis has been the reaction of ketosteroid isomerase (KSI). Studies of the binding of phenolates to KSI have been taken as proof that the electrostatic preorganization effect only makes a minor contribution to the binding of the real, multiring, transition state (TS). However, our simulation study has determined that the difference between the phenolates and the TS arises from the fact that the nonpolar state of the phenolate can rotate freely relative to the oxyanion hole and thus loses the preorganization contribution. A recent study explored the reactivity of both small and multiring systems and concluded that their similar reactivity contradicts our preorganization idea. Herein, we establish that the available experiments in fact provide what is perhaps the best proof and clarification of the preorganization idea and its crucial role in enzyme catalysis. First, we analyze the binding energy and the pK(a) of equilenin and identify direct experimental evidence for our prediction about the differential electrostatic stabilization of the large TS and the small phenolates. Subsequently, we show that the similar reactivity of the small and large systems is also due to an electrostatic preorganization effect but that this effect only appears in the intermediate state because the TS is not free to rotate. This establishes the electrostatic origin of enzyme catalysis. We also clarify the crucial importance of having a well-defined physical concept when examining catalytic effects and the need for quantitative tools for analyzing such effects.

Fig. 1.

A schematic description of the isomerization catalyzed by KSI.

Fig. 2.

The binding configuration of equilenin. Note that in this illustration, the residue in position 40 is an aspartate. However, experimental studies of the binding of equilenin have been performed with not only Asp40 but also a D40N mutation.

Fig. 3.

The binding energies (including the relevant electrostatic contribution, highlighted in black) for the systems being considered in this work. The figure also depicts the charging step in the binding cycle for equilenin and the IS of S_mini.

Fig. 4.

The energetics of the reactions of S_mini, S_full^′, and S_full. The relevant energies are taken form the analysis in Figs. S5 and S6. The figure also illustrates the preorganization effect in the charging of S_full and the IS of S_mini. In the case of equilenin, the uncharged state of the IS is fixed so that the preorganization energy is large and negative. In the case of S_mini, the uncharged state of the IS is free to rotate, leading to a very small preorganization contribution.

Fig. 5.

The nature of the electrostatic energy of the TS and IS of the reaction of S_mini. The figure presents the result of the FEP charging process for each system in water and in the protein and explains why the difference between the charging in the protein and in water is larger for the TS. As seen from the figure, the uncharged TS is not free to rotate and thus has a significant preorganization contribution, whereas the uncharged state of the IS is free to rotate in the active site.

Fig. 6.

The change in distance between the oxygen of Tyr16, and the oxygen of the IS of S_mini during simulations where the protein sees zero charge on the IS. As seen from the figure, the uncharged state of the IS is free to rotate in the active site.