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

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.