Subject Paper No and Title 16 Bio-organic and Biophysical Module No and Title 22 Mechanism of Enzyme Catalyzed reactions I Module Tag CHE_P16_M22 Chymotrypsin
TABLE OF CONTENTS 1. Learning outcomes 2. Introduction 3. Structure of Chymotrypsin 4. Mechanism of action of Chymotrypsin 4.1 Kinetics of Chymotrypsin enzyme action 4.2 Production of Active Chymotrypsin takes place via proteolytic cleavage from an inactive precursor. 5. Chymotrypsin and related proteases 6. Summary
1. Learning Outcomes After studying this module you shall be able to: Learn the structure of Chymotrypsin Learn the kinetics of action of Chymotrypsin Learn Mechanism of action of Chymotrypsin 2. Introduction Chymotrypsin an enzyme that belongs to the general class of serine proteases. Chymotrypsin is a proteolytic enzyme acting in the digestive systems of many organisms that is produced and secreted by the pancreas. It facilitates the cleavage of peptide bonds by a hydrolysis reaction, a process that, albeit thermodynamically favorable, occurs extremely slowly in the absence of a catalyst. Chymotrypsin catalyzes the reaction rate by a factor of 10 9. Chymotrypsin preferentially cleaves peptide amide bonds where the carboxyl side of the amide bond (the P 1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine).these amino acids contain an aromatic ring in their side chain that fits into a 'hydrophobic pocket' (the S 1 position) of the enzyme. 3. Structure of Chymotrypsin Chymotrypsin is a globular protein of 245 amino acid residues and has a Molecular Weight of 25,000. It consists of three polypeptide chains A, B and C that are linked via disulfide bonds. There are a total of five disulfide bonds holding these three polypeptide chains together. These three chains are held together by two inter-chain disulfide bonds. There also three intra-chain disulfide bonds. The chains run between amino acids 1-13 (chain A), 16-146 (chain B), and 149-245 (chain C). The five disulfide bonds are between cysteine side-chains at positions 1 and 122, 42 and 58, 136 and 201, 168 and 182, and 191 and 220.These disulfide bonds are crucial to the attainment of the correct secondary structure of Chymotrypsin i.e. to the correct folding of the protein. Chymotrypsin is folded into two domains (amino acids 27-112 and 133-230) each consisting of six beta strands arranged as antiparallel beta sheets in order to form a beta barrel. The overall shape of the molecule is that of an ellipsoid with a maximum dimension of 5.1 nm. The appropriate folding of this protein is closely linked to its action on its substrate. There is a shallow depression at the active site in which the side chains, Ser 195, His 57, and Asp 102 which are important for its catalytic activity are embedded. The three amino acid residues that participate in substrate binding at the enzyme active site are far away in the primary sequence of the enzyme but are brought in close proximity in the crevice between the two protein domains.
4. Mechanism of action of Chymotrypsin Fig.1 3D structure of chymotrypsin 4.1 Kinetics of Chymotrypsin enzyme action: The kinetics of the reaction catalyzed by Chymotrypsin was first elucidated by the experiments of B.S. Hartley and B.A. Kilby in 1954: Hartley and Kilby showed that the hydrolysis of peptide bonds by Chymotrypsin takes place in two stages, an initial burst phase at the beginning of the reaction which then levels off to a steady-state phase following Michaelis-Menten kinetics. Thus, there is a formation of a covalently bound enzyme substrate intermediate. Fig.2 Kinetics of chymotrypsin catalysis
It is also called "ping-pong" mechanism. The cleavage of the peptide bond by Chymotrypsin is a two step hydrolysis reaction i.e. it is an addition of a water molecule. First acylation of the substrate takes place to form an acyl-enzyme intermediate. This is followed by a deacylation step wherein hydrolysis of the acyl intermediate is accompanied by regeneration of the enzyme. The active site of Chymotrypsin, marked by serine 195, lies in a cleft on the surface of the enzyme. The enzyme action occurs via the concerted action of the three amino acid residues in the catalytic triad. These three amino acids are serine 195, Histidine 57 and Aspartate 102. Placement of these amino acids in a linear array forms what is known as the catalytic triad (Fig3.). The linear arrangement of these three amino acid residues allows for a charge relay to take place from Aspartate to Serine leading to activation of the Serine195 residue. Fig3. The catalytic triad
Fig4. Ser protease mechanism Step 1: When substrate (polypeptide) binds, the side of chain of the residue next to the peptide bond to be cleaved nestles in a hydrophobic pocket on the enzyme, positioning the peptide bond for attack. In the substrate binding pocket of the enzyme, Histidine 57 extracts one proton from serine to form an alkoxide ion. This serine ion reacts with the substrate. Step 2: The carboxylate R-group of Asp102 forms a hydrogen bond with N-δ hydrogen of His 57, increasing the pka of its ε nitrogen and thus making it able to deprotonate serine 195. This deprotonation of Ser195 by His57 turns it into a strong nucleophile that can now attack the substrate. Oxygen develops a partially negative charge in the oxyanion hole.
Fig5. Oxyanion hole Oxygen develops a partially negative charge in the oxyanion hole. The serine side chain now binds to the electron-deficient carbonyl carbon of the protein main chain. Ionization of the carbonyl oxygen is stabilized by formation of two hydrogen bonds to adjacent main chain N-hydrogens. This occurs in the oxyanion hole. Oxyanion hole stabilizes the tetrahedral intermediate. It is formed by hydrogen bonds linking peptide NH groups to the negatively charged oxygen atom. Step 3: Instability of the negative charge on the substrate carbonyl oxygen when will lead to collapse of the tetrahedral intermediate, re-formation of a double bond with carbon which breaks the peptide bond between the carbon and amino acid group. The amino leaving group is protonated by His57, facilitating its displacement. The leaving group is stabilized and the acylenzyme enzyme intermediate, bound to the serine, is formed. Step 4: The newly formed amino terminus of the cleaved protein now dissociates and binds to Serine. This completes the first stage (acylation of enzyme). The first product has been made. Step 5: In the second reaction step, a water molecule is activated by the basic Histidine, and acts as a nucleophile.. Histidine 57 deprotonates the water to form a strongly nucleophilic hydroxide ion. Attack of hydroxide ion on the ester linkage of the acylenzyme generates a second tetrahedral intermediate. This hydroxide group attaches to carbon from the carboxy side and destabilizes the acyl intermediate.
Step 6: The oxygen of water attacks the carbonyl carbon of the serine-bound acyl group, resulting in formation of a second tetrahedral adduct. Step 7: Collapse of the tetrahedral intermediate forms the second product, a carboxylate anion, and displace Ser195. The proton from Histidine goes back to Serine regenerating the serine -OH group. Step 8: The carboxylic acid is released and the enzyme is reformed and made available for next reaction in the cell. Chymotrypsin also hydrolyzes other amide bonds in peptides at slower rates, particularly those containing Leucine at the P 1 position. Fig6. Detailed mechanism The Asp.His.Ser charge relay system is also present in the zymogen. However, the zymogen is not enzymatically active because the substrate-binding pocket is not properly formed. During activation, the cleavage of the Arg 15- Ile 16 bond by Trypsin creates a new positive charge at the α-amino group of Ile 16. A strong electrostatic force between this positive charge and that of the side chain of Asp 194 helps to move parts of the molecule, such as the side-chains of Arg 145 and Met 192, and to form the substrate binding pocket. The electrostatic interaction between Ile 16 and Asp 194 is strong because it occurs in a region of low dielectric constant in the interior of the enzyme.
4.2. Production of Active Chymotrypsin takes place via proteolytic cleavage from an inactive precursor. Chymotrypsin is produced by the acinar cells of the pancreas as an inactive precursor (zymogen) known as Chymotrypsinogen. The unusual structure of Chymotrypsin arises as a result of activation of the zymogen by proteolytic cleavage. The activation of Chymotrypsin occurs in stages. First, there is an initial cleavage by Trypsin, of the Arg-15 Ile 16 bond that yields a fully active, two chain, enzyme known as Π-chymotrypsin. Subsequent cleavage catalysed by Chymotrypsin of the Leu13-Ser 14 bond then takes place to yield δ-chymotrypsin. Finally, α-chymotrypsin is generated by the autocatalytic cleavage of the Tyr 146- Thr 147 and Asn 148- Ala149 bonds. (fig.) α-chymotrypsin is the most widely studied form of the enzyme. There are two isoforms of pancreatic Chymotrypsin A and B, which are known to cleave proteins selectively at specific peptide bonds formed by the hydrophobic residues tryptophan, phenylalanine and tyrosine. Fig7. Activation of chymotrypsin 4. Chymotrypsin and related proteases The serine proteases Chymotrypsin, Trypsin and Elastase possess very similar three dimensional structures, but display quite different specificities for substrates. Chymotrypsin is specific for amides with aromatic or other large hydrophobic side-chains, Trypsin is specific for amides with positively charged side-chains (Lys or Arg), and Elastase has a somewhat broader specificity. The
difference in specificity strongly correlates with the differences in the substrate-binding pockets of the three enzymes. While in Chymotrypsin there is a hydrophobic interaction between the substrate and non polar side chains in the active site, in Trypsin, the active site has an aspartic acid that interacts with the positively charged side-chain of the substrate. The active site of Elastase although is very similar to that of Chymotrypsin but its obstructed by the bulky side chains of Val 216 and Thr 226 which makes the active site inaccessible to potential substrates. Another example of a serine protease that uses a similar mechanism to hydrolyze peptide bonds is Subtilisin. It is a 245 amino acid globular protein with several alpha helices and a large beta sheet. It is structurally unrelated to Chymotrypsin but uses the similar catalytic triad in its substrate binding pocket. This is a classic example of convergent evolution. 5. Inhibition of Chymotrypsin and related proteases Chymotrypsin is both an esterase and proteolytic enzyme. Both these enzymatic activities are inhibited by diisopropyl fluorophosphonate (DFP). A common substrate used to experimentally study the enzymatic activity of Chymotrypsin is para nitrophenyl ester as the hydrolysis product of this substrate can be conveniently assayed spectrophotometrically. 6. Summary Chymotrypsin is a digestive enzyme that is secreted by the acinar cells of the pancreas and acts in the small intestine to hydrolyze the proteins. It s a globular protein with significant alpha helical and beta sheet content. Chymotrypsin belongs to a class of serine proteases because Serine 195 is the main amino acid in the active site of the enzyme. The active site of Chymotrypsin is lined with non polar amino acid residues and thus it favorably accommodates large hydrophobic amino acid side chains in its binding pocket. Chymotrypsin specifically cleaves the peptide bond at the carboxyl side of tryptophan, Tyrosine and Phenylalanine..Chymotrypsin is synthesized in the pancreas as an inactive precursor or zymogen. Successive proteolytic cleavages lead to formation of the active chymotrypsin enzyme. The Asp 102.His 47 Ser 195 catalytic triad is the main component of the active site. The linear arrangement of these amino acid residues is such that the Serine residue is deprotonated and makes electrostatic interaction with the peptide bond. Chymotrypsin follows the ping pong mechanism. The cleavage of the peptide bond by Chymotrypsin proceeds via an acyl intermediate.
Other digestive enzymes that are structurally related to Chymotrypsin and use a similar mechanism of action include Elastase and Trypsin. Subtilisn is another member of the serine protease family that shares the same catalytic triad mechanism of action to hydrolyze the peptide bonds but is not structurally related to Chymotrypsin