Untitled

Enzyme Mechanisms II

March 26, 2026

Lecture Notes

  • Understand what lysozyme does.

  • Know the reaction of lysozyme.

  • Know how a serine protease works.

  • Understand how different enzymes choose their substrate.

  • Understand what affinity labeling of an enzyme is.

Lecture Outline

  1. Lysozyme

  2. Serine Proteases

  3. Serine Proteases Catalytic Triad

Lysozyme

Mechanism of Lysozyme

  • Lysozyme Function: Digests bacterial cell walls by breaking β(1-4) glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG). This breakdown releases structural integrity from bacterial cells, aiding the immune response.

Lysozyme Structure

  • Amino acid sequence of Lysozyme includes multiple residues: Leu, Asp, Asn, Tyr, Arg, Gly, Ser, among others. Notably, positions such as 35 (Glu) and 52 (Asp) are crucial for enzymatic activity.

  • Key Residue Positions:

    • 35: Glu

    • 52: Asp

    • 195: Ser

Lysozyme Structure Continued

  • Relevant structures include:

    • Amino end of chain

    • Pleated sheet region

    • Substrate cleavage site

    • Hydrogen bond interaction between Glu and the substrate

    • Disulfide bridge exists between key structural amino acids.

Lysozyme Interaction with Substrate

  • Maximum activity observed when substrate (bacterial polysaccharides) binds effectively within the enzyme’s binding cleft, further resulting in product formation.

Rates of Lysozyme-Catalyzed Hydrolysis

TABLE 14-2: Rates of Hydrolysis for Selected Oligosaccharide Substrates

Compound

kcat (S^-1)

(NAG)₂

2.5 × 10^-8

(NAG)₃

8.3 × 10^-6

(NAG)₄

6.6 × 10^-5

(NAG)₅

0.033

(NAG)₆

0.25

  • The activation of lysozyme follows a structured sequence that begins with the binding of the substrate, ultimately leading to a hydrolytic reaction that releases oligosaccharides.

The Phillips Mechanism

  • Steps in Reaction:

    1. Lysozyme attaches to a bacterial cell wall by binding to a hexasaccharide unit, distorting the D residue towards a half-chair conformation.

    2. Glu 35 transfers its proton to the O1 of the D ring via general acid catalysis; the C1-O1 bond is cleaved, generating an oxonium ion at C1.

    3. Asp 52 stabilizes the oxonium ion through charge-charge interactions. Due to distance, it cannot form a covalent bond directly with the substrate.

    4. The E ring group is released from the enzyme, yielding a glycosyl-enzyme intermediate which then reacts with water to reverse the chemistry and reprotonate Glu 35.

Schematic of Lysozyme Reaction

  • Mechanism demonstrates general acid-base catalysis and covalent intermediates involved in substrate hydrolysis.

Intermediate Step Trap

  • NAG₂FGlcF serves as a transition state trap stabilizing the oxoanion intermediate. Detected as a covalent attachment to Asp 52, captured via X-ray crystallography.

Serine Proteases

TABLE 14-4: Selection of Serine Proteases

Source

Enzyme

Function

Pancreas

Trypsin

Digestion of proteins

Pancreas

Chymotrypsin

Digestion of proteins

Pancreas

Elastase

Digestion of proteins

Blood

Thrombin

Blood clotting

Blood

Plasmin

Dissolution of blood clots

Various

Kallikrein

Control of blood flow

Various

Complement C1

Immune response

Various

Acrosomal protease

Penetration of ovum

Various

Lysosomal protease

Cell protein turnover

Various

Cocoonase

Dissolution of cocoon after metamorphosis

Various

α-Lytic protease

Possibly digestion

Various

Proteases A and B

Possibly digestion

Streptomyces

Subtilisin

Possibly digestion

Active Serine Presence in Proteases

  • Active Serine (Ser195): A covalent bond between Serine 195 and the substrate indicates catalytic activity. This serine can be inhibited by agents such as diisopropyl fluorophosphate (DIPF), which specifically inactivates the enzyme.

  • Due to the potential toxicity of inhibition, nerve gases (e.g., sarin) and certain insecticides act by similar mechanisms impacting Serine residues crucial for neuromuscular function.

Affinity Labeling

  • His 57 represents a crucial catalytic residue.

  • Involves a substrate with a reactive group binding at the enzyme’s active site, inducing a reaction with nearby reactive amino acids—analogous to a Trojan horse effect.

  • TPCK (Tosyl-L-phenylalanine chloromethyl ketone) as a model for studies involving affinity labeling.

Serine Proteases Catalytic Triad

  1. Active Site Residues:

    • Asp 102

    • His 57

    • Ser 195

    • Gly 193

Protease Mechanism Overview

  • The catalytic activity is noted through key steps, beginning with substrate binding to Ser195, which subsequently adopts a nucleophilic role in attacking peptide bonds, leading to the formation of tetrahedral intermediates. His 57 operates as a proton donor, managing acid-base catalysis throughout the process.

Detailed Kinetics Observations

  1. Burst Phase: Rapid formation of product correlating stoichiometrically with enzyme.

  2. Steady State: Characterized by slower reaction rates independent of substrate concentrations.

Triad Charge Transfer Complex Stabilization

  • The interactions between His 57 and Asp 102 create a charge relay system facilitating stabilization of tetrahedral intermediates during substrate processing.

Conclusion

  • Enzyme dynamics, specifically in lysozyme and serine proteases, illustrate intricate mechanisms of action and the evolutionary parallels among various enzymes. Understanding these mechanics can inform biochemistry, therapeutic advancements, and pivotal ecological roles.