Lecture 6

Enzymes as Catalysts

  • Overview

    • Presented by Dr. Martin Wiese, The University of Strathclyde

    • Based on 'Introduction to Nutrition and Metabolism' and 'BM210 Introduction to Biochemistry'

    • Covers enzyme function, activity, specificity, and inhibitors.

Learning Outcomes

  • Understand the function of the active site of enzymes

  • Understand the role of cofactors in enzyme function

  • Know about enzyme activity and specificity

  • Understand the factors affecting enzyme activity

  • Understand enzyme inhibition

Part A: Enzyme Function

  • Enzymes as Catalysts

    • Enzymes lower the activation energy required for a reaction, thus accelerating chemical reactions.

    • Enzymes are not consumed during the reaction and do not affect the chemical equilibrium.

    • Catalysis Essence: Specific stabilization of the transition state:

    • Reactants [A] + [B] yield products [C] + [D].

Properties and Structure of Enzymes

  • Enzymes are proteins, large organic compounds made from amino acids.

  • The primary structure (linear sequence) folds into a tertiary structure (3D shape).

    • This folding creates an active site where substrates bind.

Stages of an Enzyme-Catalyzed Reaction

  • Reaction sequence:

    1. Substrates (Enz + S)

    2. Forming the enzyme-substrate complex (Enz-S)

    3. Conversion to products (Enz-P)

    4. Release of the product (Enz + P)

    • Weak interactions hold substrates in the active site.

    • After the reaction, the active site can bind new substrates.

Active Site Properties

  • The active site's properties include:

    • Correct positioning of substrates for optimal reaction orientation.

    • Complementarity with the transition state formed during the reaction.

    • Stabilization of the transition state, lowering activation energy, and increasing reaction rate.

    • The transition state comes together rapidly to form products that bind less tightly and are released from the enzyme.

Interactions in the Active Site

  • Non-covalent interactions between substrates and amino acid side chains include:

    • Acidic Groups: Aspartate (Asp), Glutamate (Glu) → ionic bonds.

    • Basic Groups: Lysine (Lys), Histidine (His), Arginine (Arg) → ionic bonds.

    • Hydrophilic Interactions:

    • Hydroxyl groups (Serine, Threonine, Tyrosine)

    • Thiol groups (Cysteine)

    • Amide groups (Asparagine, Glutamine)

    • Aromatic Interactions: Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp)

    • Hydrophobic Interactions: Alanine (Ala), Leucine (Leu), Isoleucine (Ile), Valine (Val), Methionine (Met)

Catalytic Mechanisms

  • Reactive Groups in Enzymes at catalytic sites catalyze reactions by:

    • Donating or withdrawing electrons

    • Stabilizing or generating free radical intermediates

    • Forming temporary covalent bonds with substrates.

Enzyme Specificity

  • Enzymes are highly specific and catalyze only one type of reaction.

  • Shape, Charge, and Conformation of substrates are crucial for enzyme binding.

Classification of Enzymes

  1. Oxidoreductases: Catalyze oxidation and reduction reactions, e.g., dehydrogenases.

  2. Transferases: Transfer functional groups, e.g., kinases.

  3. Hydrolases: Catalyze hydrolysis of various bonds.

  4. Lyases: Add or remove groups from double bonds.

  5. Isomerases: Catalyze isomerization reactions.

  6. Synthetases: Synthesize bonds between substrates using ATP.

Measurement of Enzyme Activity

  • Enzyme Activity Units:

    • Defined as the amount of substrate converted in mmol/min under specific conditions (30°C).

    • 1 enzyme unit (EU) = 1 μmol min^-1

  • Specific Activity: Activity per mg of total protein (μmol min^-1mg^-1); reflects the enzyme's purity.

Reaction Rate Characteristics

  • Reaction rate is measured by the formation of products over time at fixed enzyme concentration, temperature, and pH.

  • Reaction rate exhibits a hyperbolic curve due to:

    • Accumulation of product

    • Depletion of substrate

    • Possible enzyme denaturation

Key Takeaway

  • For accurate enzyme activity assays, ensure measurement of initial velocities (V0).

Key Terms from Part A

  • Enzyme specificity

  • Classes of enzymes and reactions

  • Specific activity

  • Rate of reaction

Part B: Factors Affecting Enzyme Activity

Main Factors

  1. pH

  2. Temperature

  3. Concentration of enzyme

  4. Concentration of substrate

  5. Covalent modification of enzyme

Effect of pH

  • pH measures the acidity/alkalinity of a solution:

    • Neutral = pH 7.00

    • Acidic < 7.00; Basic > 7.00.

  • Enzyme Activity vs. pH:

    • Ionization state of amino acid side chains is influenced by pH, affecting enzyme function.

    • Example: Acid phosphatase vs. alkaline phosphatase performance varies with pH.

Effect of Temperature

  • Higher temperatures increase reaction rates due to faster molecular movement and collision frequency. However:

    • Denaturation can occur if temperatures are too high, leading to loss of enzyme activity.

    • Optimum temperature will depend on incubation time.

Effect of Enzyme Concentration

  • Predictable linear increase in product formation with increasing enzyme concentration.

    • Active dimers may dissociate at low concentrations, and monomers may exhibit low or no activity.

Substrate Concentration Dependence

  • Higher substrate concentrations lead to a reaction rate approach to Vmax, with Km defined as the concentration of substrate required to reach half Vmax.

  • Michaelis-Menten Equation: v=V<em>max[S]K</em>m+[S]v = \frac{V<em>{max}[S]}{K</em>m + [S]}

    • Describes the steady-state rate of reaction concerning substrate concentration.

Km: The Michaelis Constant

  • High Km indicates low affinity for substrate; low Km indicates high affinity.

    • Enzymes with low Km compared to substrate concentration ([S]) operate at maximum rates.

  • Typical Km values:

    • Chymotrypsin: 5 mM

    • Pyruvate Carboxylase: 60 µM

    • Protein Kinase: 12 µM

Relevance of Km

  • Example of competitive enzyme advantage; with lower Km, one enzyme can outperform the other when substrate concentration is low.

Experimental Determination of Km and Vmax

  • Determine by incubating the enzyme under optimal conditions and evaluating a range of substrate concentrations.

    • Lineweaver-Burk plot helps estimate Km and Vmax from reaction rate data:

    • Plot obtained by reciprocal rates and substrate concentrations:
      1v=K<em>mV</em>max⋅1[S]+1Vmax\frac{1}{v} = \frac{K<em>m}{V</em>{max}} \cdot \frac{1}{[S]} + \frac{1}{V_{max}}

Enzyme Reactions with Multiple Substrates

Types of Reactions

  1. Sequential Reaction: Each substrate binds in order, forming a ternary complex (A-Enz-B).

  2. Ping-pong Reaction: One substrate modifies the enzyme before the second binds.

Cooperative Binding and Allosteric Enzymes

  • Allosteric Regulation: Multi-subunit complexes demonstrate cooperative binding where substrate binding at one site enhances binding at others.

Covalent Modification of Enzymes

  • Modifications such as phosphorylation can induce significant structural changes leading to altered activity.

Key Terms from Part B

  • Factors affecting enzyme activity

  • Michaelis-Menten equation

  • Ternary complex

  • Ping-pong reaction

  • Allosteric regulation

  • Cooperativity

  • Covalent modification

Part C: Enzyme Inhibitors

Types of Inhibitors

  • General Effects:

    • Decrease enzyme's ability to bind substrates.

    • Reduce enzyme's catalytic activity.

Reversible vs. Irreversible Inhibitors

  • Reversible Inhibitors: Bind non-covalently, can be competitive or non-competitive.

  • Irreversible Inhibitors: Bind covalently, contributing to permanent enzyme inactivation.

Competitive Inhibitors

  • Compete with substrates for binding to the active site,

    • Functionality depends on concentrations of substrate and inhibitor.

  • Effects of Competitive Inhibition:

    • Vmax remains unchanged; Km increases with higher inhibitor concentrations.

Mixed Inhibitors

  • May increase or decrease Km, decrease Vmax consistently.

  • Do not bind at the active site but distort substrate binding sites affecting reaction kinetics.

Non-competitive Inhibitors

  • Bind separate from active site, not competing with substrates.

    • Effects: Km remains unchanged; Vmax decreases with increasing inhibitor concentration.

Allosteric Inhibitors

  • Change enzyme conformation leading to increased Km and reduced activity due to lower substrate affinity.

Key Terms from Part C

  • Modes of inhibition and effects on rate of reaction

  • Reversible versus irreversible inhibitors

  • Competitive inhibitors

  • Mixed inhibitors

  • Non-competitive inhibitors

  • Allosteric regulation