Enzymes tetrahedral intermediated

Transition-state stabilization in chymotrypsin also involves the side chains of the substrate. The side chain of the departing amine product forms stronger interactions with the enzyme upon formation of the tetrahedral intermediate. When the tetrahedral intermediate breaks down (Figure 16.24d and e), steric repulsion between the product amine group and the carbonyl group of the acyl-enzyme intermediate leads to departure of the amine product. 

FIGURE 24.17 The mechanism of the thiolase reaction. Attack by an enzyme cysteine thiolate group at the /3-carbonyl carbon produces a tetrahedral intermediate, which decomposes with departure of acetyl-CoA, leaving an enzyme thioester intermediate. Attack by the thiol group of a second CoA yields a new (shortened) acyl-CoA. 

Ester hydrolysis is common in biological chemistry, particularly in the digestion of dietary fats and oils. We ll save a complete discussion of the mechanistic details of fat hydrolysis until Section 29.2 but will note for now that the reaction is catalyzed by various lipase enzymes and involves two sequential nucleophilic acyl substitution reactions. The first is a trcinsesterificatiori reaction in which an alcohol gioup on the lipase adds to an ester linkage in the tat molecule to give a tetrahedral intermediate that expels alcohol and forms an acyl... 

The tetrahedral intermediate expels a diacylglycerol as the leaving group and produces an acyl enzyme. The step is catalyzed by a proton transfer from histidine to make the leaving group a neutral alcohol. 

Q The enzyme active site contains an aspartic acid, a histidine, and a serine. First, histidine acts as a base to deprotonate the -OH group of serine, with the negatively charged carboxylate of aspartic acid stabilizing the nearby histidine cation that results. Serine then adds to the carbonyl group of the triacylglycerol, yielding a tetrahedral intermediate. 

The tetrahedral intermediate expels the serine as leaving group in a second nucleophilic acyl substitution reaction, yielding a free fatty acid. The serine accepts a proton from histidine, and the enzyme has now returned to its starting structure. 

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. 

As with an isolated double bond, epoxide formation in an aromatic ring, i.e., arene oxide formation, can occur mechanistically either by a concerted addition of oxene to form the arene oxide in a single step, pathway 1, or by a stepwise process, pathway 2 (Fig. 4.78). The stepwise process, pathway 2, would involve the initial addition of enzyme-bound Fe03+ to a specific carbon to form a tetrahedral intermediate, electron transfer from the aryl group to heme to form a carbonium ion adjacent to the oxygen adduct followed by... 

Fig. 3.6. Stereoelectronic control of the cleavage of the tetrahedral intermediate during hydrolysis of a peptide bond by a serine hydrolase. The thin lines represent the reactive groups of the enzyme (serine, imidazole ring of histidine) the thick lines represent the tetrahedral intermediate of the transition state. The full circles are O-atoms open circles are N-atoms. The dotted lines represent H-bonds the thick double arrow indicates an unfavorable dipole-dipole interaction [21]. A (R)-configured N-center B (S)-configured N-center.

Even lower temperatures have been used to study possible intermediate stages in the formation of the acyl enzyme. A tetrahedral intermediate (with a covalent bond between the substrate carbonyl carbon atom and the oxygen atom of the active site serine) (Fig. 2) had been suggested by analogy with nonenzymatic reactions. With rapid reaction techniques, spectrophotometric evidence has been obtained for an additional intermediate before the acyl enzyme in the case of chromophoric substrates. By using first the protein fluorescence emission (Fink and Wildi, 1974)... 

Fig. 2. The generally accepted mechanism for the hydrolysis of peptide substrates by the serine proteases. The precise locations of the protons are still moot their positions here are taken from Steitz and Shullman (1982). I, Michaelis complex II and V, tetrahedral intermediates III and IV, acyl-enzyme VI, product complex.

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

The mechanism of catalysis by these enzymes has been extensively investigated (for review see ref. 10). Essentially, the active site serine via its side chain hydroxyl group performs a nucleophilic attack on the carbonyl carbon of the scissile peptide bond thus forming a tetrahedral intermediate. A histidine residue in the active site serves as a general base accepting the proton from the serine residue. The acyl enzyme thus formed is broken down via a nucleophilic attack of a water molecule to complete the hydrolysis of the peptide bond. 

All of this evidence supports the existence of tetrahedral intermediates in a-chymotrypsin-catalysed reactions, but it should be noted that O-exchange with water is not observed in deacylation of cinnamoyl- 0-chymotrypsin, in contrast with the hydrolysis of O-cinnamoyl-N-acetylserinamide where such exchange is detected (Bender and Heck, 1967). Lack of exchange in the enzyme reaction could reflect interactions of the tetrahedral intermediate with the protein. 

Using a rapid quench-fiow kinetic assay for post-complex fragment formation, Nair and Cooperman showed that the ET encounter complex of serpin and enzyme forms both E I and the post-complex fragment with the same rate constant, indicating that both species arise from ET conversion to E I. These results support the conclusions (a) that the peptide bond remains intact within the ET complex, and (b) that E I is likely to be either the acyl-enzyme or the tetrahedral intermediate formed after water attack on acyl-enzyme. 

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