Proteases and Catalysis

Chapter 9 Study Questions 

1. Four major catalytic strategies. 
2. Draw the catalytic triad in chymotrypsin. 
3. What are the tetrahedral intermediates in the reaction cycle of chymotrypsin? 
4. What is the oxyanion hole in chymotrypsin? 
5. Difference between serine proteases, cysteine proteases, aspartyl proteases, and metal protease.  Give one example for each group. 
6. Draw the catalytic center of carbonic anhydrase II. 
7a. What is pKa of H2O in the catalytic center of carbonic anhydrase II?

7b. What is pKa of H2O in pure water? 
8. What are the functions for Zn(II) in carbonic anhydrase II? 
9. Buffer and pH (homework example). 
10. What are the differences between three types of carbonic anhydrases? 
11. Define convergent evolution and divergent evolution. 
12. The relation between restriction and modification processes of DNA.  
13. Structure of the catalytic center in restriction enzyme EcoRV. 
14. Interaction of cognate DNA and non-cognate DNA with restriction enzyme. 

Catalytic Strategies

  • Covalent Catalysis

    • Definition: Formation of covalent intermediates during the catalytic process.

    • Mechanism: Involves the temporary bonding of the substrate to the catalyst, effectively transforming the substrate into a more reactive form.

  • Acid-Base Catalysis

    • Definition: Utilization of molecules other than water as proton donors or acceptors to facilitate the reaction.

    • Importance: Adjusts the pH environment to enhance reactivity, often involving key amino acid side chains in the active site of the enzyme.

  • Metal Ion Catalysis

    • Statistic: At least one-third of enzymes incorporate metal ions in their structure.

    • Function: Metal ions can stabilize negative charges, participate in oxidation-reduction reactions, and interact with substrates to promote catalysis.

  • Catalysis by Approximation

    • Definition: The strategy of bringing two substrates in close proximity to increase the likelihood of a reaction occurring.

    • Mechanistic Role: Enhances the effective concentration of reactants, which can substantially increase reaction rates.

Examples of Catalytic Enzymes

  • Proteases

  • Carbonic Anhydrases

  • Restriction Enzymes

Proteases

  • Functions

    • Critical role in the ingestion and digestion systems.

    • Involved in blood coagulation processes.

    • Facilitate programmed cell death (apoptosis).

Mechanism of Proteases

  • Peptide Bonds:

    • Definition: Peptide bonds are highly stable chemical bonds that link amino acids in proteins.

    • Activity: Proteases cleave these bonds rapidly, typically within milliseconds.

Structure of Chymotrypsin

Three chains, therefore it is a quaternary structure. Count the N-terminals in order to determine the subunits of the structure. 3 N-terminals = more than one subunit.

  • Chemical Representation:

    • Visual depiction of chymotrypsin structure including functional groups:

    • Involves components like H3C, NH, N, C, and varying side chains including Ala (Alanine), Phe (Phenylalanine), Asn (Asparagine), Ser (Serine), Met (Methionine), Glu (Glutamic acid) alongside Phe, Trp (Tryptophan), and Tyr (Tyrosine), Met.

Kinetics of Chymotrypsin

  • Steady-state Phase:

    • Monitoring of absorbance related to p-nitrophenol released during the reaction.

    • Characterized by a burst phase occurring milliseconds after substrate mixing.

Inhibition of Chymotrypsin

  • Irreversible Inhibitor:

    • Example: Diisopropylphosphofluoridate (DIPF).

    • Structure includes Ser 195, depicting binding and activity inhibition.

Catalytic Triad in Chymotrypsin

  • Key Amino Acids

    • Serine 195, Histidine 57, and Aspartate 102 play critical roles within the catalytic mechanism.

    • The catalytic triad acts as a nucleophile enhancing the protease's functionality.

    • Serine can donate Hydrogen to Histidine. This would make Histidine the donor. This allows for functionality in different pH environments.

    • The Alkoxide ion on serine has the ability to catalyze the reaction in order to cleave peptide bonds.

Steps of Chymotrypsin Protease:

Step 1: Binding of the substrate to the protease's active site occurs, positioning the peptide bond for optimal attack by the nucleophile.

Nucleophilic Attack Mechanism

Step 2: Tetrahedral Intermediate with Oxyanion hole:

  • Description of nucleophilic attack where the nucleophile donates both bonding electrons to the electrophile, leading to a tetrahedral intermediate formation.

  • Key components include the oxyanion hole, which stabilizes the tetrahedral intermediate, facilitating the reaction pathway.

Acyl-Enzyme Complex Formation

Step 3: Peptide bond is cleaved. Amino end stays with histidine via hydrogen bonding. Carboxyl group is covalently bound with Serine 195:

  • Progression to the acyl-enzyme state involves the transient bonding of the enzyme to the substrate's acyl component.

  • Step 4: amino terminal is released

  • Step 5: The reaction is hydrolyzed. Hydrogen bond forms between Ser 195 and His 57.

  • Step 6: The second tetrahedral intermediate is formed.

    • Oxyanion hole contributes in stabilizing the structure.

  • Step 7: Carboxyl end is Released

  • Step 8: Recovery

Variations of Oxyanion Hole in Different Proteases

  • Example: Ser 221, Asp 32, Asn 155, His 64 observed in subtilisin (originating from Bacillus amyloliquefaciens).

Specificity of Chymotrypsin and Other Proteases

If you cleave one peptide bond, you may create new reactive sites that can further influence the specificity of other proteases, leading to a more complex interaction network.

  • Chymotrypsin Specificity:

    • Key residues at positions Trp 215, Gly 216, Gly 226, define substrate specificity.


  • Specificity of Other Serine Proteases:

    • Proteases like Trypsin, with unique amino acid compositions, show differential binding preferences.

    • Asp 189: negative charge attracts the basic amino acid residues.

Cysteine Proteases

  • Definition: Cysteine acts instead of serine, activated by histidine to fulfill the role of the nucleophile.

  • Example: Papain, isolated from papaya.

Caspases as Cysteine Proteases

  • Involved in apoptosis, with multiple components such as Caspase 9, Granzyme B, and various substrates including PARP (poly(ADP-ribose) polymerase) and Lamins being cleaved during cellular processes.

Aspartyl Proteases

  • Examples: Renin

    • Importance: Crucial for regulating blood pressure.

  • HIV Protease:

    • Aspartyl protease plays a vital role in HIV replication; essential for the activation of viral proteins.

Specificity of HIV Protease and Its Inhibitors

  • Inhibitors are tailored to bind specifically to HIV protease to prevent viral replication.

Cocktail Drugs for HIV Patients

  • Integrated treatment regimens include:

    • Entry inhibitors (CCR5 receptor antagonists), Reverse transcriptase inhibitors, Protease inhibitors, Integrase inhibitors, and Maturation inhibitors.

Metalloproteases

  • Examples: Carboxypeptidase A, matrix proteases.

  • Metal ion (e.g., Zn2+) plays a critical role in enzymatic activity and substrate interaction.

Summary of Proteases

  • Types:

    • Serine or Threonine Proteases: Chymotrypsin, Trypsin, Elastase, Subtilisin.

    • Cysteine Proteases: Papain, Caspases.

    • Aspartic Proteases: Renin, HIV proteases.

    • Metalloproteases: Carboxypeptidase, matrix proteases.