Oxyanion with tetrahedral intermediates

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

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... 

A water molecule is bound in the oxyanion hole of proteinase K, a member of the subtilisin family (Betzel et al., 1988). Its replacement by the carbonyl oxygen of the substrate is suggested to be concerted with formation of the tetrahedral intermediate. 

As opposed to the catalytic triad, which is made up of side chains that can now be mutated at will, the structure-function relationships in the oxyanion hole are not equally susceptible to experimental verification. Only in subtilisin the involvement of the side-chain amide of Asn-155 allows for quantitadve assessment of the role of the oxyanion H bonds in the stabilization of the tetrahedral intermediates. The replacement of Asn-155 with an isosteric Leu reduces the Acat by 200- to 300-fold, but leaves the Km essentially unaffected (Bryan et al., 1986). This observation is fully consistent with Asn-155 not contributing to substrate binding, but playing a key role in the stabilization of the intermediate. 

Fig. 6. A stereo view of the structure of the oxyanion hole in RmL, as inferred from the structure of the complex with n-hexyl phosphonate ethyl ester. The dotted lines represent the two hydrogen bonds to the tetrahedral intermediate.

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