Biochemistry - The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Types of enzyme-catalyzed reactions
Enolic intermediates in isomerization reactions
∆5-3-Ketosteroid isomerase and other enzymes catalyzing 1,3-proton migration

In animal Tissues, Cholesterol serves as the precursor for all Steroid Hormones, including such 3-ketosteroids as progesterone and testosterone (Ch. 12, Sect. E.3). While cholesterol contains a double bond at the 5,6-position—as seen in the compound on the left in equation (7-56)—in ketosteroids this double bond is conjugated with a carbonyl group. First, the hydroxyl group of cholesterol is oxidized to a keto group (step a). This is followed by a virtually irreversible migration of the double bond catalyzed by ∆⁵-3-ketosteroid isomerase (step b) [137]. This enzyme has been isolated in a fully purified state from Pseudomonas testosteroni. It is an oligomeric protein with a Molecular Weight of ~40,000, possessing an exceptionally high molecular activity (~10⁵ s^-1) largely driven by an unusually low value of ∆H^≠. The protein is highly hydrophobic and soluble in media containing high concentrations of ethanol. In the case of the animal-derived enzyme, this property indicates that the protein is localized within The Endoplasmic reticulum.

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Substrates containing ²H at the 4-position react at a rate that is only one-fourth that of normal substrates. The large isotope effect indicates that the Cleavage of the C—H bond leading to The formation of an enolate anion (presumably enzyme-stabilized) is the rate-determining step (step a in the scheme below):

The axial proton at C-4 is abstracted [138] and must be transferred by a group on the enzyme molecule to the 6-position, from which it is returned to the substrate, again into an axial position (step b), demonstrating a syn-transfer. No solvent proton exchange is observed during this reaction, presumably due to the exceptionally high catalytic turnover rate of the isomerase. That the reaction indeed involves H⁺ transfer rather than an alternative mechanism is supported by the following observation: with poor substrates and Competitive Inhibitors such as nortestosterone [which contains a double bond in the same position as the reaction product in (7-57)], one of the hydrogen atoms at C-4 undergoes complete exchange with the medium. Furthermore, the UV absorption spectrum of the inhibitor undergoes a shift from 248 to 258 nm upon interaction with the enzyme. This finding strongly suggests the formation of a stabilized enolate anion as depicted in scheme (7-57).

The enzyme-catalyzed isomerization of cis-aconitate to trans-aconitate [equation (7-58)] also appears to involve proton transfer [139]:

Whether Resonance in the anion extends to the carboxyl groups remains unclear, though it is reasonable to suppose that it does and that the isomerization mechanism is likely analogous to that of the ketosteroid isomerase-catalyzed reaction. However, other allylic rearrangements exist where the substrates are compounds lacking either carbonyl or carboxyl groups. It remains unknown whether these rearrangements proceed via anionic or carbonium ion intermediates.

Allylic rearrangements involving proton transfers may also constitute an integral part of the mechanism in other Types of Enzyme-Catalyzed Reactions.