Oxyanion Holes with Enolate Intermediates

Typically, the hydrogen bond donor for the tetrahedral intermediate oxyanion is the amide NH group. In contrast, in enolate oxyanion holes, a much more variable range of hydrogen bond donors is observed, such as water, a tyrosine hydroxyl. 

In addition, many oxyanion holes have been discovered in which water is one of the hydrogen bonding donors. These waters are clearly defined in the respective electron density maps. In some enzymes these waters are part of a trail of water molecules [80, 81]. When water is used as the hydrogen bond donor, very often the intermediate that is stabilized is not the tetrahedral intermediate but an enolate, which is derived by base-catalyzed deprotonation of the Ca-atom of the 

There are only few experimental studies where the importance of the oxyanion hole for stabilizing the enolate oxyanion has been quantified. Tonge and collaborators [21] have studied the properties of the Glyl41Pro variant of hydratase. In this variant, one of the two peptide NH groups of the oxyanion hole (N(Glyl41)) has been disabled because of the Glyl41Pro mutahon. Compared to the wild-type enzyme, kcat is reduced 10 -fold, whereas is not affected. In addition, Raman studies suggest that the active site of the variant remains intact. 

Detailed binding with substrate and transition state analogs has also been reported on KSI [83, 84] using a wide range of techniques, highlighting the subtle interplay of the electrostatic and geometric properties of the enolate stabilizing active site. 

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Enzymes with oxyanion holes are now known to catalyze a wide range of reactions with substrates that have a carbonyl moiety. The examples discussed in this chapter include thioesters, oxygen esters, peptides, and ketones (Figure 4.1). Two classes of high-energy intermediates with oxyanions are generated in these reactions (Table 4.3), a tetrahedral intermediate and an enolate. These reactions are... 

The final step involves the attack on the acyl-thioester by the enolate, with the tetrahedral intermediate stabilised by the oxyanion hole. The fi-keto product is then formed following collapse of the tetrahedral intermediate. The elongated intermediate is now free to diffuse out of the active site, albeit thethered to the receiving ACP, and downstream for further modification. 

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