Chymotrypsin's Catalytic Mechanism: Videos & Practice Problems

Chymotrypsin's catalytic mechanism consists of two phases: acylation and deacylation. In the acylation phase, substrate binding occurs, followed by a nucleophilic attack by serine 195, forming a tetrahedral intermediate stabilized by the oxyanion hole. This leads to the cleavage of the peptide bond and the formation of an acyl enzyme via a covalent ester linkage. The deacylation phase involves water acting as a nucleophile, regenerating the original enzyme by hydrolyzing the ester bond. This mechanism highlights the importance of covalent catalysis and enzyme-substrate interactions in biochemical reactions.

Chymotrypsin's Catalytic Mechanism

Chymotrypsin is a digestive enzyme that plays a crucial role in the cleavage of peptide bonds, particularly those involving aromatic amino acids. Its catalytic mechanism can be divided into two distinct phases: the acylation phase and the deacylation phase. Understanding these phases is essential for grasping how chymotrypsin functions at a molecular level.

The acylation phase is characterized by the formation of a covalent bond between the enzyme and the substrate. This phase begins with the active site of chymotrypsin, which contains a catalytic triad, prominently featuring the serine residue. The catalytic triad enhances the nucleophilicity of serine, allowing it to effectively attack the peptide bond of the substrate. During this phase, the peptide bond is cleaved, resulting in the formation of an acyl-enzyme intermediate, where part of the substrate remains covalently attached to the enzyme.

Following the acylation phase, the deacylation phase aims to regenerate the original enzyme. This is achieved through the addition of water, which hydrolyzes the acyl-enzyme bond, releasing the remaining portion of the substrate. The catalytic triad once again plays a vital role, as it facilitates the removal of a hydrogen atom, restoring serine's nucleophilic properties. This regeneration allows chymotrypsin to repeat the catalytic cycle, enabling it to cleave additional peptide bonds.

In summary, chymotrypsin's catalytic mechanism involves a two-phase process: acylation, where the enzyme forms a covalent bond with the substrate, and deacylation, where the enzyme is restored to its original state. This intricate mechanism highlights the enzyme's efficiency and specificity in peptide bond cleavage.

Chymotrypsin's Catalytic Mechanism

Chymotrypsin's catalytic mechanism begins with the acylation phase, which can be divided into four key steps: substrate binding, nucleophilic attack, removal of the leaving group, and the conclusion of the phase. The primary focus of this phase is the formation of a covalent ester linkage between chymotrypsin's active site and the substrate, which is a peptide that chymotrypsin cleaves.

In the first step, substrate binding occurs when the peptide, characterized by an amino end and a carboxyl end, binds to the active site of chymotrypsin. The active site contains a catalytic triad composed of aspartate 102, histidine 57, and serine 195. The peptide bond that chymotrypsin targets for cleavage is located closest to the carboxyl end, specifically involving the aromatic amino acid phenylalanine, which is recognized by the enzyme.

The second step involves nucleophilic attack, where serine 195, established as a strong nucleophile through hydrogen bonding with histidine 57 and aspartate 102, attacks the carbonyl carbon of the substrate. This interaction leads to the formation of an unstable tetrahedral intermediate. The oxyanion hole within the active site stabilizes this intermediate, allowing the reaction to proceed.

In the third step, the tetrahedral intermediate collapses, facilitated by histidine 57 acting as an acid. This results in the cleavage of the peptide bond, releasing the amine portion of the substrate. The electron density from the peptide bond is utilized to facilitate this reaction, leading to the formation of the conjugate base of histidine.

The final step marks the end of the acylation phase, where the enzyme is now covalently linked to the substrate through the ester linkage, forming what is known as the acyl enzyme. The released amine portion of the substrate diffuses away, leaving the enzyme in its acylated state, ready to transition into the subsequent deacylation phase, which will restore the enzyme to its original form.

Overall, the acylation phase of chymotrypsin's mechanism highlights the importance of covalent catalysis in forming an ester linkage and breaking the targeted peptide bond, setting the stage for further enzymatic action.

Chymotrypsin's Catalytic Mechanism

The deacylation phase of chymotrypsin's catalytic mechanism follows the acylation phase and consists of four key steps that restore the enzyme to its original state, allowing it to catalyze another reaction. This phase primarily involves the hydrolysis of the covalent ester linkage formed during the acylation phase, which is a specific type of catalytic mechanism known as ester hydrolysis.

In the first step of deacylation, a water molecule (H₂O) enters the active site of chymotrypsin. This marks the beginning of the deacylation process. The second step involves nucleophilic attack, where histidine 57 acts as a base, deprotonating the water molecule to generate a hydroxide ion. This hydroxide ion then attacks the carbonyl carbon of the substrate, forming a tetrahedral intermediate. The stability of this intermediate is enhanced by the oxyanion hole within the active site, which stabilizes the negative charge that develops during this transition.

The third step is the removal of the leaving group. The tetrahedral intermediate collapses, with histidine 57 now acting as an acid, donating a proton to facilitate the cleavage of the ester bond. This step results in the breaking of the ester linkage, allowing the carboxylic acid portion of the substrate to be released. Finally, in the fourth step, the deacylation phase concludes with the regeneration of the original chymotrypsin enzyme, now ready to engage in another catalytic cycle.

Overall, the deacylation phase mirrors the acylation phase in its structure, emphasizing the cyclical nature of chymotrypsin's catalytic activity. The enzyme's ability to undergo these phases efficiently is crucial for its role in catalyzing peptide bond hydrolysis in proteins.

Chymotrypsin's acylation phase is below. The catalytic process of __________ is illustrated by arrow # ____ and the catalytic process of _________________________ is illustrated by arrow # ____.

• A. General-Base catalysis; 1 ; covalent catalysis; 2.
• B. General-Acid catalysis; 1 ; covalent catalysis; 3.
• C. Covalent catalysis; 2 ; covalent catalysis; 3.
• D. Covalent catalysis; 1 ; General-Base catalysis; 3.

Answer: (A)

Chymotrypsin's Catalytic Mechanism

Chymotrypsin is a serine protease that catalyzes the hydrolysis of peptide bonds through a two-phase mechanism: acylation and deacylation. The catalytic triad of chymotrypsin consists of three key amino acids: aspartate 102, histidine 57, and serine 195, which work together to facilitate the reaction.

In the acylation phase, the process begins with the binding of the substrate to the active site of chymotrypsin. This is followed by a nucleophilic attack where histidine 57 acts as a base, facilitating the conversion of serine 195 into a stronger nucleophile. The hydroxyl group of serine 195 attacks the carbonyl carbon of the substrate, leading to the formation of a tetrahedral intermediate. This intermediate is stabilized by the oxyanion hole within the active site. The next step involves the collapse of this tetrahedral intermediate, where histidine 57 donates a proton, resulting in the cleavage of the peptide bond and the release of the first product. This leaves a covalent acyl-enzyme complex, which is crucial for the subsequent deacylation phase.

During the deacylation phase, a water molecule enters the active site, and histidine 57 again acts as a base, extracting a proton from water to generate a hydroxide ion. This hydroxide ion performs a nucleophilic attack on the carbonyl carbon of the acyl-enzyme complex, forming another tetrahedral intermediate, which is again stabilized by the oxyanion hole. The final step involves the collapse of this intermediate, leading to the cleavage of the ester bond and the release of the second product. Ultimately, the active site is regenerated, allowing chymotrypsin to catalyze another reaction cycle.

This intricate mechanism highlights the importance of the catalytic triad and the stabilization of intermediates, showcasing how enzymes like chymotrypsin efficiently facilitate biochemical reactions.

In the mechanism of chymotrypsin, serine-195:

• A. Functions as a nucleophile.
• B. Functions as an electrophile.
• C. Forms a tetrahedral intermediate with part of the substrate.
• D. Is reduced by Aspartate 102.
• E. Is oxidized by Histidine 57.
• F. A and C.
• G. A, C and E.

Answer: (F)

Sequentially number the following steps of Chymotrypsin's catalytic mechanism in the correct order from 1-8:
a) Released newly formed amine portion of the substrate diffuses away _______
b) His-57 deprotonates the Ser-195 hydroxyl group, generating a stronger nucleophile ______
c) Tetrahedral intermediate collapses & His-57 donates a H+ to N of scissile bond (cleaved peptide bond) ______
d) Via general-base-catalysis, His-57 deprotonates a water molecule, generating OH- ______
e) Tetrahedral intermediate collapses & His-57 donates a proton to Ser-195 (cleaving ester bond) _______
f) Released newly formed carboxylic acid portion of the substrate diffuses away & enzyme is restored _______
g) Hydroxide ion attacks the carbonyl group of the substrate, forming another tetrahedral intermediate ______
h) Nucleophilic Ser-195 attacks the carbonyl C of the substrate forming a tetrahedral intermediate______

Chymotrypsin's catalytic mechanism is a complex process divided into two main phases: the acylation phase and the deacylation phase. Understanding the sequence of events in these phases is crucial for grasping how this enzyme functions. The acylation phase begins with the generation of a stronger nucleophile, which is facilitated by histidine 57 deprotonating the hydroxyl group of serine 195. This initial step, often considered step 0, sets the stage for substrate binding.

Once the stronger nucleophile is established, serine 195 performs a nucleophilic attack on the carbonyl carbon of the substrate, leading to the formation of a tetrahedral intermediate. This is a critical step in the acylation phase, as it allows for the subsequent removal of the leaving group. The tetrahedral intermediate then collapses, with histidine 57 donating its acidic hydrogen to the nitrogen of the peptide bond being cleaved, resulting in the release of the newly formed amine portion of the substrate. This marks the end of the acylation phase.

Transitioning into the deacylation phase, the next step involves substrate binding again, where histidine 57 deprotonates a water molecule that enters the active site, generating a hydroxide ion. This hydroxide ion then performs a nucleophilic attack on the carbonyl group of the substrate, forming another tetrahedral intermediate. Following this, the tetrahedral intermediate collapses, with histidine 57 donating its acidic proton to serine 195, cleaving the ester bond. Finally, the released carboxylic acid portion of the substrate diffuses away, restoring the chymotrypsin enzyme to its original state, ready to catalyze another reaction.

In summary, the steps of chymotrypsin's catalytic mechanism can be ordered as follows: 1) histidine 57 deprotonates serine 195, 2) serine 195 performs a nucleophilic attack, 3) removal of the leaving group occurs, 4) the acylation phase ends, 5) histidine 57 deprotonates a water molecule, 6) the hydroxide ion attacks the carbonyl group, 7) removal of the leaving group in the deacylation phase, and 8) the deacylation phase concludes with the release of the carboxylic acid.

Here’s what students ask on this topic:

What are the two phases of chymotrypsin's catalytic mechanism?

Chymotrypsin's catalytic mechanism consists of two phases: the acylation phase and the deacylation phase. In the acylation phase, the substrate binds to the enzyme's active site, and serine 195 performs a nucleophilic attack on the carbonyl carbon of the substrate, forming a tetrahedral intermediate. This intermediate is stabilized by the oxyanion hole, leading to the cleavage of the peptide bond and the formation of an acyl-enzyme complex via a covalent ester linkage. In the deacylation phase, a water molecule acts as a nucleophile, hydrolyzing the ester bond to regenerate the original enzyme, allowing it to catalyze another reaction.

How does the catalytic triad in chymotrypsin function?

The catalytic triad in chymotrypsin consists of three amino acids: aspartate 102, histidine 57, and serine 195. These residues work together to enhance the nucleophilicity of serine 195. Aspartate 102 stabilizes histidine 57 through hydrogen bonding, making histidine a better base. Histidine 57 then deprotonates serine 195, converting it into a strong nucleophile. This nucleophilic serine 195 can then attack the carbonyl carbon of the substrate, forming a tetrahedral intermediate and facilitating peptide bond cleavage.

What role does the oxyanion hole play in chymotrypsin's catalytic mechanism?

The oxyanion hole in chymotrypsin's active site plays a crucial role in stabilizing the tetrahedral intermediate formed during the catalytic process. When serine 195 attacks the carbonyl carbon of the substrate, the resulting tetrahedral intermediate is unstable. The oxyanion hole, a region in the active site, stabilizes this intermediate by forming hydrogen bonds with the negatively charged oxygen atom. This stabilization is essential for the intermediate to form and proceed to the next steps of the catalytic mechanism, ultimately leading to peptide bond cleavage.

What is the significance of covalent catalysis in chymotrypsin's mechanism?

Covalent catalysis is a key feature of chymotrypsin's catalytic mechanism. During the acylation phase, serine 195 forms a covalent ester linkage with the substrate, creating an acyl-enzyme intermediate. This covalent bond is crucial for the cleavage of the peptide bond. In the deacylation phase, water acts as a nucleophile to hydrolyze this ester bond, regenerating the original enzyme. Covalent catalysis allows for a more controlled and efficient cleavage of peptide bonds, highlighting the enzyme's specificity and catalytic power.

How does water contribute to the deacylation phase in chymotrypsin's mechanism?

In the deacylation phase of chymotrypsin's catalytic mechanism, water acts as a nucleophile. A water molecule enters the active site and is deprotonated by histidine 57, forming a hydroxide ion. This hydroxide ion then performs a nucleophilic attack on the carbonyl carbon of the acyl-enzyme intermediate, forming a tetrahedral intermediate. The oxyanion hole stabilizes this intermediate, allowing it to collapse and break the ester bond. This hydrolysis reaction regenerates the original enzyme, making it ready for another catalytic cycle.