Exam 2 Lecture 3

Chapter 1: Introduction to Chymotrypsin Mechanism

  • Overview of chymotrypsin mechanism

    • Focus on Chapter 6 of the study material

    • Discuss the experimental approaches biochemists use to understand enzyme mechanisms

    • Reminder:

    • Problem set 2 due tonight

    • Quiz on Monday and Tuesday (based on section schedule)

  • Characteristics of Chymotrypsin

    • Class: Protease (enzyme that cleaves peptide bonds)

    • Action: Cleaves amide bonds on C-terminal side of large hydrophobic amino acids

    • Reaction notes:

    • Thermodynamically favorable but with high activation energy

    • Requires enzymes to enhance the reaction rate

    • Rate enhancement by chymotrypsin: at least 10910^9 fold

  • Experimental categories to be discussed:

    1. Effect of pH on catalysis

    2. Use of irreversible inhibitors

    • Definition: Form covalent bond with the enzyme, permanently inactivating it

    • Two classes to evaluate

    1. Pre-steady state kinetics

    • Involvement of a chromogenic substrate (yellow at basic pH)

    • Detected via absorbance spectroscopy

    1. Structural studies and homology analysis

Chapter 2: pH Dependence on Catalysis

  • Experiment examining the pH rate profile for chymotrypsin

    • Observed: Bell-shaped pH rate profile

    • Analysis conclusion:

    • Two amino acids play roles in the catalysis

      1. Amino acid with pKa around 7 (needs to be deprotonated for catalysis)

      2. Amino acid with pKa around 9 (needs to be protonated for maximal catalysis)

Chapter 3: Irreversible Inhibitors as Probes of the Active Site

  • Use of irreversible inhibitors to understand enzyme activity

    • General characteristic of irreversible inhibitors:

    • Form covalent complexes with the enzyme

Example: DIPF

  • Structure: Diisopropyl phosphofluoridate

  • Type: Group specific reagent

    • Targets: Specific functional group (alcohol group of serine)

  • Mechanism of action:

    • Forms covalent bond with serine residue in chymotrypsin

    • Modification result: 1 out of 28 serines in chymotrypsin modified (indicates critical reactive site)

Affinity Labels or Substrate Analogs

  • Definition: Resemble substrate structure and contain a chemically reactive group

  • Example in another context: Bromoacetyl phosphate for triosephosphate isomerase

  • Application with Chymotrypsin:

    • Affinity label used: Tosyl phenylalanine chloromethyl ketone (TPCK)

  • Conclusion from experiments:

    • Identifies the important histidine in the active site for catalysis

Chapter 4: Reactive Group Analysis

  • Designing new affinity labels for chymotrypsin

    • Need for two components: substrate-like structure and reactive group

  • Examples of potential changes for affinity labels:

    • Substituting amino acid side chains (e.g., using isoleucine, methionine, or tryptophan)

    • Changing the reactive group to another electrophile like bromide or an aldehyde due to their high reactivity

Chapter 5: Pre-Steady State Kinetics

  • Description of pre-steady state kinetics

    • Rapid mixing of substrate and enzyme, reaction quenched within milliseconds

    • Observations related to types of substrates

    • Use of esters instead of amides accelerates reaction detection

  • Result conclusions:

    • Burst phase kinetics indicative of covalent catalysis

    • Mechanism involves initial covalent bond formation between enzyme and substrate, followed by hydrolysis event

    • Catalytic mechanism implies formation of a covalent enzyme intermediate

Chapter 6: High-Resolution Structural Studies

  • Analysis of highly conserved amino acids across species

    • Important amino acids identified:

    • Aspartate

    • Histidine

    • Serine

    • Glycine

  • Role of Catalytic Triad:

    • Serine as a nucleophile activated by histidine, which is polarized by aspartate

    • Formation of oxyanion hole by glycine for tetrahedral intermediate stabilization

  • Overview of the complete mechanism of chymotrypsin:

    • Active site dynamics explain the high catalytic efficiency (rate enhancing factor of 10910^9)

Conclusion and Key Summary Points

  • The catalytic triad involves:

    • Serine (nucleophile)

    • Histidine (activator)

    • Aspartate (polarizing agent)

  • Chymotrypsin's function is intricately linked to structural aspects and specific amino acid interactions, forming the basis for its catalytic mechanism and efficiency in proteolytic activity.