Tetrahedral oxyanion intermediate

The probable mechanism of action of chymotrypsin. The six panels show the initial enzyme-substrate complex (a), the first tetrahedral (oxyanion) intermediate (b), the acyl-enzyme (ester) intermediate with the amine product departing (c), the same acyl-enzyme intermediate with water entering (d), the second tetrahedral (oxyanion)... 

Scheme 11.1 General acid and base catalysis of deamidation. The tetrahedral oxyanion intermediate is inferred to be the transition state.

F. 8.8. Chymotrypsin hydrolyzes certain peptide bonds in proteins. The scissile bond is shown in blue. The carbonyl carbon, which carries a partial positive charge, is attacked by a hydroxyl group from water. An unstable tetrahedral oxyanion intermediate is formed, which is the transition state complex. As the electrons return to the carbonyl carbon, it becomes a carboxylic acid, and the remaining proton from water adds to the leaving group to form an amine. 

The formation of the oxyanion intermediate during serine protease action is also supported by the existence of tetrahedral forms of enzymes inhibited by substratelike aldehydes. The -OH group of Ser 195 can add to the carbonyl group to form hemiacetals. For example, a 13C-enriched aldehyde whose carbonyl carbon had a chemical shift of 204 ppm gave a 94 ppm resonance as it formed the tetrahedral hemiacetal with one of the inhibitory aldehydes, N-ace tyl-i -Len-i-Leu-L-arginal... 

Figure 11.6 A schematic view of the presumed binding mode of the tetrahedral transition state intermediate for the deacylation step. The four essential features of the serine proteinases are highlighted in yellow the catalytic triad, the oxyanion hole, the specificity pocket, and the unspecific main-chain substrate binding.

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. 

From the first transition state (TSl, Fig. 1), the reaction path leads to the tetrahedral intermediate 1 (INTI). In the latter, the proton transfer from methanol to the tertiary amine function is completed (from 1.183 to 1.059 A), and the negative charge at the former carbonyl oxygen atom reaches its maximum. This charge is compensated by a further shortening of the bifurcated hydrogen bonds to 2.040 A (-0.103 A) and 1.765 A (-0.096 A) (Fig. 1). The thiourea moiety thus forms an oxyanion hole similar to the amide groups of the serine protease backbone [41]. 

The importance of oxyanion holes in enzymes was first discovered in chymot-rypsin by David Blow and co-workers and in subtihsin by Jo Kraut and co-workers [39]. In these enzymes, a tetrahedral intermediate is generated after nucleophilic attack of a deprotonated serine side chain on the peptide carbonyl group. It was recognized from the begirming that the geometry of the active site complexes was possibly better complementary to the tetrahedral intermediate than to the planar peptide substrate [40]. 

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... 

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