Chymotrypsin, acyl-enzyme intermediate

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. 

Chymotrypsin catalysis takes place through a three-step process, equation (11), where ES is an enzyme substrate complex which breaks down to give an acylated enzyme intermediate, ES and Pj,... 

Chymotrypsin enhances the rate of peptide bond hydrolysis by a factor of at least 109. It does not catalyze a direct attack of water on the peptide bond instead, a transient covalent acyl-enzyme intermediate is formed. The reaction thus has two distinct phases. In the acylation phase, the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme. In the deacylation phase, the ester linkage is hydrolyzed and the nonacylated enzyme regenerated. 

FIGURE 6-19 Pre-steady state kinetic evidence for an acyl-enzyme intermediate. The hydrolysis of p-nitrophenylacetate by chymotrypsin is measured by release of p-nitrophenoi (a colored product). Initially, the reaction releases a rapid burst of p-nitrophenol nearly stoichiometric with the amount of enzyme present. This reflects the fast acylation phase of the reaction. The subsequent rate is slower, because enzyme turnover is limited by the rate of the slower deacylation phase. 

MECHANISM FIGURE 6-21 Hydrolytic cleavage of a peptide bond by chymotrypsin. The reaction has two phases. In the acylation phase (steps to ), formation of a covalent acyl-enzyme intermediate is coupled to cleavage of the peptide bond. In the deacylation phase (steps to ), deacylation regenerates the free enzyme this is essentially the reverse of the acylation phase, with water mirroring, in reverse, the role of the amine component of the substrate. Chymotrypsin Mechanism... 

In addition to participating in acid-base catalysis, some amino acid side chains may enter into covalent bond formation with substrate molecules, a phenomenon that is often referred to as covalent catalysis.174 When basic groups participate this may be called nucleophilic catalysis. Covalent catalysis occurs frequently with enzymes catalyzing nucleophilic displacement reactions and examples will be considered in Chapter 12. They include the formation of an acyl-enzyme intermediate by chymotrypsin (Fig. 12-11). Several of the coenzymes discussed in Chapters 14 and 15 also participate in covalent catalysis. These coenzymes combine with substrates to form reactive intermediate compounds whose structures allow them to be converted rapidly to the final products. 

Acyl-enzyme intermediates. Serine proteases are probably the most studied of any group of enzymes.229 Early work was focused on the digestive enzymes. The pseudosubstrate, p-nitrophenyl acetate, reacts with chymotrypsin at pH 4 (far below the optimum pH for hydrolysis) with rapid release of p-nitro-phenol and formation of acetyl derivative of the enzyme. 

Steps in the hydrolysis of p-nitrophenyl acetate by chymotrypsin. In the hydrolysis of this and most other esters, the breakdown of the acyl-enzyme intermediate is the rate-determining step. In the hydrolysis of peptides and amides, the rate-determining step usually is the formation of the acyl-enzyme intermediate. This makes the transient formation of the intermediate more difficult to study because the intermediate breaks down as rapidly as it forms. 

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)... 

Titration of the intact active site obviates problems due to inactive protein which contribute to a false molarity. Active-site titrations of acyl group transfer enzymes such as a-chymotrypsin utilise a substrate which has a good leaving group. This enables the buildup of an acyl enzyme intermediate which forms faster than it can degrade and results in... 

With regard to the use of protease in the synthetic mode, the reaction can be carried out using a kinetic or thermodynamic approach. The kinetic approach requires a serine or cysteine protease that forms an acyl-enzyme intermediate, such as trypsin (E.C. 3.4.21.4), a-chymotrypsin (E.C. 3.4.21.1), subtilisin (E.C. 3.4.21.62), or papain (E.C. 3.4.22.2), and the amino donor substrate must be activated as the ester (Scheme 19.27) or amide (not shown). Here the nucleophile R3-NH2 competes with water to form the peptide bond. Besides amines, other nucleophiles such as alcohols or thiols can be used to compete with water to form new esters or thioesters. Reaction conditions such as pH, temperature, and organic solvent modifiers are manipulated to maximize synthesis. Examples of this approach using carboxypeptidase Y (E.C. 3.4.16.5) from baker s yeast have been described.219... 

Efficient modification steps through the proper orientation of the inhibitor reactive group to the enzyme nucleophile is realized by covalent bond formation. A classic example of this type is the modification of a methionine residue of chymotrypsin by /7-nitrophenyl bromoacetyl a-aminoisobutyrate (26)47). In this instance, the reactive group (bromoacetyl) is fixed at the locus near the active site through a covalent bond by means of acyl enzyme intermediates. 

Three of the four pancreatic proteases (trypsin, chymotrypsin, and elastase) are called serine proteases because they are all dependent for activity on the side chain of a serine residue in the active site. This serine residue attacks the carbonyl group of the peptide bond to cleave the peptide, giving an acyl-enzyme intermediate (Chap. 8). This ester bond is then hydrolyzed in a second step ... 

More than a third of all known proteolytic enzymes are serine proteases (2). The family name stems from the nucleophilic serine residue within the active site, which attacks the carbonyl moiety of the substrate peptide bond to form an acyl-enzyme intermediate. Nucleophilicity of the catalytic serine is commonly dependent on a catalytic triad of aspartic acid, histidine, and serine—commonly referred to as a charge relay system (3). First observed by Blow over 30 years ago in the structure of chymotrypsin (4), the same combination has been found in four other three-dimensional protein folds that catalyze hydrolysis of peptide bonds. Examples of these folds are observed in trypsin. 

Figure 9.5. Covalent Catalysis. Hydrolysis by chymotrypsin takes place in two stages (A) acylation to form the acyl-enzyme intermediate followed by (B) deacylation to regenerate the free enzyme.

This represents direct evidence for the existence of a presumably covalent acyl-enzyme intermediate in the a-chymotrypsin-catalyzed hydrolysis of a specific substrate (Fink, 1973b). 

In most cases, it is best to employ a temperature near or below the catalyst s thermostable limit (determined from the technical hterature, the manufacturer, or through screening experiments). However, in a few cases, low temperature operahon is desired. One example is acyl transfer reachons involving pephde. A common problem associated with this category is the occurrence of a side reachon, hydrolysis. Moreover, a compehhon exists between the amino acid acyl acceptor and water, both acting as nucleophiles toward the acyl-enzyme intermediate. Adlercreutz and co-workers discovered that the selectivity of chymotrypsin toward acyl exchange over hydrolysis increased as temperature decreased. - Hence, operation is best at lower temperatures. A second situation is when low temperature selectively precipitates the desired product (discussed below). 

Serine carbohydrate esterases and transacylases. The commonest reaction mechanism is the standard serine esterase /protease mechanism, demonstrated paradigmally for chymotrypsin, involving an acyl-enzyme intermediate. The enzyme nucleophile is a serine hydroxyl, which is hydrogen bonded the imidazole of a histidine residue, whose other nitrogen is hydrogen bonded to a buried, but ionised, aspartate residue (Figure 6.28),... 

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