Chymotrypsin covalent catalysis

CHYMOTRYPSIN FRUCTOSE-2,6-BISPHOSPHATASE ILLUSTRATE COVALENT CATALYSIS... 

Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway. 

Figure 3-2. Reaction mechanism of chymotrypsin as an example of covalent catalysis. Step I involves attack of the enzyme s active site serine on the peptide bond to be cleaved. In step II, a covalent complex is formed between the enzyme and a portion of the substrate (peptide 2) with release of the rest of the substrate (peptide I). Step III involves hydrolysis of the enzyme-substrate complex, which releases peptide 2 and completes the reaction.

Most enzymes employ a combination of several catalytic strategies to bring about a rate enhancement. A good example of the use of both covalent catalysis and general acid-base catalysis is the reaction catalyzed by chymotrypsin. The first step is cleavage of a peptide bond, which is accompanied by formation of a covalent linkage between a Ser residue on the enzyme and part... 

Bovine pancreatic chymotrypsin (Mr 25,191) is a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds. This protease is specific for peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr). The three-dimensional structure of chymotrypsin is shown in Figure 6-18, with functional groups in the active site emphasized. The reaction catalyzed by this enzyme illustrates the principle of transition-state stabilization and also provides a classic example of general acid-base catalysis and covalent catalysis. 

Chymotrypsin is a serine protease with a well-understood mechanism, featuring general acid-base catalysis, covalent catalysis, and transition-state stabilization. 

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. 

Covalent catalysis. In covalent catalysis, the active site contains a reactive group, usually a powerful nucleophile that becomes temporarily covalently modified in the course of catalysis. The proteolytic enzyme chymotrypsin provides an excellent example of this mechanism (Section 9.1.2). 

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.

A number of proteolytic enzymes participate in the breakdown of proteins in the digestive systems of mammals and other organisms. One such enzyme, chymotrypsin, cleaves peptide bonds selectively on the carboxyl-terminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine (Figure 9.1). Chymotrypsin is a good example of the use of covalent catalysis. The enzyme employs a powerful nucleophile to attack the unreactive carbonyl carbon atom of the substrate. This nucleophile becomes covalently attached to the substrate briefly in the course of catalysis. 

Explain why a burst of product appears when chymotrypsin reacts "with a chromogenic ester substrate, and relate this phenomenon to covalent catalysis. 

Finally an interesting report on catalysis of peptide-bond formation using a-chymotrypsin covalently bound to silica has appeared results are similar to those obtained with the free enzyme. 

The mechanism of action of anticholinesterases is to form a stable covalent complex with the Achase enzyme. Achase is one of several enzymes known as serine esterases. Other examples include the intestinal enzymes trypsin and chymotrypsin as well as the blood clotting agent thrombin. During the course of the catalysis the alcohol -OH of a serine side chain in the active site of the enzyme forms an ester complex, called the acyl-enzyme, with the substrate. So, acetylcholine will go through similar chemical reactions with Achase. 

A reaction looked at earlier simulates borate inhibition of serine proteinases.33 Resorufin acetate (234) is proposed as an attractive substrate to use with chymotrypsin since the absorbance of the product is several times more intense than that formed when the more usual p-nitrophcnyl acetate is used as a substrate. The steady-state values are the same for the two substrates, which is expected if the slow deacylation step involves a common intermediate. Experiments show that the acetate can bind to chymotrypsin other than at the active site.210 Brownian dynamics simulations of the encounter kinetics between the active site of an acetylcholinesterase and a charged substrate together with ah initio quantum chemical calculations using the 3-21G set to probe the transformation of the Michaelis complex into a covalently bound tetrahedral intermediate have been carried out.211 The Glu 199 residue located near the enzyme active triad boosts acetylcholinesterase activity by increasing the encounter rate due to the favourable modification of the electric field inside the enzyme and by stabilization of the TS for the first chemical step of catalysis.211... 

Figure 9.8. Peptide Hydrolysis by Chymotrypsin. The mechanism of peptide hydrolysis illustrates the principles of covalent and acid-base catalysis. The dashed green lines indicate favorable interactions between the negatively charged aspartate residue and the positively charged histidine residue, which make the histidine residue a more powerful base.

Finally, a molecule of water participates in the chemistry of catalysis. The HO" group attacks the covalently bound part of the substrate (dietary protein), resulting in its discharge fmm the chymotrypsin molecule (Figure 2.WF). The H of water then binds to Ser 195, regenerating the serine group to its iriitial non-ionized state. 

FIGURE 6-10 Covalent and general acid-base catalysis. The first step in the reaction catalyzed by chymotrypsin is the acylation step. The hydroxyl group of Ser is the nucleophile in a reaction aided by general base catalysis (the base is the side chain of His ). This provides a new pathway for the hydrolytic cleavage of a peptide bond. Catalysis occurs only if each step in the new pathway is faster than the uncatalyzed reaction. The chymotrypsin reaction is described in more detail in Figure 5-21. 

---