Enzymes Study Notes

Raffles Institution: Enzymes - 2026-2027

CORE IDEA

  • The Cell and Biomolecules of Life - ENZYMES

Learning Outcomes

  • Candidates should be able to:

    • Explain the mode of action of enzymes in terms of:

    • Active site

    • Enzyme–substrate complex

    • Lowering of activation energy

    • Enzyme specificity using lock-and-key and induced-fit hypotheses.

    • Investigate and explain the effects of:

    • Temperature

    • pH

    • Enzyme concentration

    • Substrate concentration

    • Describe the structure of competitive and non-competitive inhibitors with reference to their binding sites.

    • Explain the effects of competitive and non-competitive inhibitors (including allosteric inhibitors) on enzyme activity.

    • Apply knowledge gained to new situations or related problems.

    • Explain the regulation of enzyme activity and its role in metabolism.

References

  • Campbell, N.A. and Reece, J.B. (2011), Biology (9th edition), Pearson Benjamin-Cummings, San Francisco.

  • Hoh, Y. K. (2002), Longman A-Level Course in Biology (Vol. I), Longman.

  • Taylor, D.J., Green, N.P.O., Stout, G.W. and Soper, R. (1997), Biological Science 1 (3rd edition), Cambridge University Press, Cambridge.

TABLE OF CONTENTS

(A) Introduction
(B) The Active Site
(C) Models of Enzyme Action

  1. Lock and Key Hypothesis

  2. The Induced Fit Model
    (D) Energy Profile of a Reaction
    (E) Enzyme Cofactors
    (F) Following the Time Course of an Enzyme-Catalysed Reaction
    (G) Factors Affecting the Rate of Enzyme-Catalysed Reactions

  3. Temperature

  4. pH

  5. Enzyme Concentration

  6. Substrate Concentration
    (H) Enzyme Inhibition

  7. Competitive Inhibition

  8. Non-Competitive Inhibition
    (I) Allosteric Regulation
    (J) Links

(A) INTRODUCTION

What Are The Roles Of Enzymes In The Cell?

  • Enzymes facilitate rapid biochemical reactions in living cells in a controlled manner.

Key Aspects

  1. Catalysis

    • Enzymes act as highly specific biological catalysts that enhance the reaction rates of metabolic reactions.

  2. Regulation

    • Provide mechanisms for controlling individual reaction rates through:

      • Allosteric control

      • Competitive inhibition

      • Non-competitive inhibition

      • Covalent modification of enzymes

      • Variation in enzyme synthesis.

    • Enzymes are crucial for life as reactions in their absence would be too slow for cellular functions.

(B) THE ACTIVE SITE

Active Site Definition and Structure

  1. Primary Structure

    • Determines secondary and tertiary structure, specifying overall 3D conformation.

  2. Active Site Characteristics

    • Small region where substrate binds, typically comprised of 3-12 amino acids.

    • Contains residues that interact via weak hydrogen and ionic bonds (contact amino acid residues).

    • Specificity arises from complementary shape and charge between substrate and active site.

    • Active site adapts shape upon substrate binding (induced fit).

  3. Roles of Amino Acids

    • Contact residues: Position substrate correctly.

    • Catalytic residues: Facilitate conversion of substrate to product.

    • Structural residues: Maintain enzyme conformation.

    • Non-essential residues: No specific function, often surface-bound.

(C) MODELS OF ENZYME ACTION

1. Lock and Key Hypothesis

  • Proposed by Fischer, 1894.

    • The active site has a specific conformation complementary to the substrate (the "key").

    • Forms enzyme-substrate (ES) complex upon binding, followed by catalysis and product formation.

    • Product release leaves the active site free for new substrate.

2. The Induced Fit Model

  • Proposed by Koshland, 1959.

    • The active site is not a perfect fit initially; substrate binding induces a conformational change for a better fit.

    • Enhances catalysis efficiency.

(D) ENERGY PROFILE OF A REACTION

Overview

  • Enzymes decrease activation energy (EA) barriers, facilitating smoother reactions.

  • Activation Energy (EA)

    • The energy required for reactants to reach the transition state.

Energy Profile Explanation

  • Enzymes do not alter the free energy ( $\Delta G$) of the reaction, but lower the EA by:

    1. Proximity effects: Increased chance of reaction due to reactants binding closely.

    2. Strain effects: Distortion of substrates increases bond reactivity.

    3. Orientation effects: Correct positioning of reactants for chemical attack.

    4. Microenvironment effects: Hydrophobic zones allow polar reactants to interact more easily.

    5. Acid-base catalysis: Amino acids in the enzyme assisting the catalytic process.

(E) ENZYME COFACTORS

Definition of Cofactors

  • Additional non-protein substances that enzymes require for catalytic activity.

Types of Cofactors

  1. Inorganic Ions

    • Example: Zinc in DNA polymerase; necessary for enzyme activation.

  2. Coenzymes

    • Organic cofactors, e.g. NAD.

  3. Prosthetic Groups

    • Permanently bound to the enzyme, e.g. haem group of cytochrome oxidase.

(F) FOLLOWING THE TIME COURSE OF AN ENZYME-CATALYSED REACTION

Methods

  1. Measure Product Formation

    • Example: Catalase decomposing hydrogen peroxide.

  2. Measure Substrate Disappearance

    • Example: Amylase digesting starch.

Experimental Procedure Example

  • For catalase:

    1. Mix catalase with hydrogen peroxide, start stopwatch.

    2. Collect evolved O2 volume via water displacement or gas syringe measurement.

    3. Plot product formation vs. time.

Trends and Interpretations

  • Volume of products formed increases initially, then slows down as substrates are depleted.

  • Determine reaction rate from the gradient of time-course graph.

(G) FACTORS AFFECTING THE RATE OF ENZYME-CATALYSED REACTIONS

1. Temperature

  • Increased temperature raises kinetic energy, leading to more effective collisions until denaturation occurs.

  • Q10 Temperature Coefficient

    • Rate doubles for every 10°C increase up to the enzyme’s optimum.

2. pH

  • Each enzyme has an optimum pH for maximal activity; deviations can lead to denaturation and decreased reactivity.

  • Changes in pH alter charge properties of amino acids, disrupting enzyme structure and function.

3. Enzyme Concentration

  • Rate of reaction correlates with enzyme concentration at low substrate availability; maximum rates plateau when saturation occurs.

4. Substrate Concentration

  • Low concentrations allow increased rates of reaction until saturation is reached (Vmax).

  • Michaelis Constant (Km)

    • The substrate concentration at ½ Vmax, indicating enzyme affinity for substrate.

(H) ENZYME INHIBITION

Types of Inhibition

  1. Competitive Inhibition

    • Inhibitor resembles substrate and competes for the active site.

    • Overcome by increasing substrate concentration.

  2. Non-Competitive Inhibition

    • Inhibitor binds to another site on the enzyme, altering its shape and decreasing its activity.

    • Cannot be overcome by increasing substrate concentration.

Comparative Summary

  • Competitive inhibitors affect Vmax, can be overcome by increased substrate.

  • Non-competitive inhibitors reduce Vmax, irreversibly alters enzyme function.

(I) ALLOSTERIC REGULATION

  • Allosteric enzymes have active sites and allosteric sites for binding regulators.

  • Can exist in two conformational states influenced by activators and inhibitors.

  • Exhibits cooperativity in substrate binding, reflected in sigmoid rate vs. [S] plots.

  • Feedback Inhibition: The end product of a metabolic pathway inhibits an earlier enzyme in the pathway, conserving resources.

(J) LINKS

Key Concepts

  • 3D conformation, active site, activation energy, allosteric site, catalytic residues, effective collisions, only catalysis, lock and key, optimum temperature, enzyme-substrate complex, induced fit.

Related Topics

  • Proteins: Many enzymes are proteins.

  • Respiration: Example involves hexokinase and NAD+.

  • DNA & Genomics: DNA polymerase and related enzymes.

  • Cell Signalling: Role of kinases and phosphatases in cellular processes.