LHYNUX BME NOTE

Enzymes And Metabolism

Introduction

• Course: Enzyme Metabolism and Bioenergetics - Cell Biology (BME 161)

• Instructor: Prince E. Adjei

• Institution: Kwame Nkrumah University of Science and Technology

• Date: 2024

Module Overview

1. Bioenergetics: The Flow of Energy in the Cell

2. Metabolic Pathways Overview

3. Enzymes: The Catalysts of Life

Learning Objectives

• Role of ATP: Explain ATP's role in bioenergetics and energy coupling in cellular processes.

• Enzyme Structure and Function: Identify enzyme structure and function, including active sites and factors influencing enzyme activity.

• Enzyme Kinetics: Analyze enzyme kinetics, focusing on enzyme inhibition and effects on metabolic reactions.

• Metabolic Pathways: Describe key metabolic pathways—glycolysis, citric acid cycle, and oxidative phosphorylation—and their role in ATP production and cellular energy metabolism.

Bioenergetics

Definition

• Bioenergetics: The branch of biochemistry dealing with energy flow and transformations in biological systems.

• It describes how living organisms acquire and transform energy to perform biological work.

• Focus on metabolic pathways is crucial for understanding bioenergetics.

Role of ATP and Energy Coupling

ATP Functionality

• Adenosine Triphosphate (ATP): The primary energy carrier in cells.

• Structure: Composed of adenine base, ribose sugar, and three phosphate groups.

• Energy Release via Hydrolysis: The reaction ATP + H₂O → ADP + Pi + Energy releases approximately ext{ΔG} ext{ ≈ } -30.5 ext{ kJ/mol} under cellular conditions.

Energy Coupling

• Energy Coupling: The process where exergonic reactions drive endergonic reactions.

• Example: The phosphorylation of glucose during glycolysis (endergonic) is coupled with ATP breakdown.

• ATP Regeneration: Roughly 10^9 ATP molecules recycled per cell per day via various metabolic pathways.

Metabolism

Definition

• Metabolism: The total of all chemical activities in cells, involved in handling material and energy resources.

• Composed of two categories:

  1. Catabolic Pathways: Break down molecules, releasing energy.

  2. Anabolic Pathways: Build complex molecules, requiring energy.

Cellular Processes

• Metabolism includes stepwise reactions, involving both energy-releasing and energy-requiring reactions.

• Cells need to perpetually produce energy to sustain ongoing chemical reactions.

Sugar Metabolism

Overview

• Sugars store energy within their bonds and are metabolized in cells to release energy.

• Glycolysis Equation: ext{C}6 ext{H}{12} ext{O}6 + 6 ext{O}2
  ightarrow 6 ext{CO}2 + 6 ext{H}2 ext{O} + ext{energy}

• Photosynthesis converts carbon dioxide to sugars while releasing oxygen: ext{6CO}2 + 6 ext{H}2 ext{O} + ext{energy}
  ightarrow ext{C}6 ext{H}{12} ext{O}6 + 6 ext{O}2

Redox Reactions and Electron Carriers

Definitions

• Redox Reactions: Chemical reactions involving electron transfer—oxidation (loss of electrons) and reduction (gain of electrons).

• Catabolic Pathways: These pathways oxidize fuels (like glucose) to release energy.

Key Electron Carriers

• NAD⁺/NADH: Accepts 2 electrons + H⁺, used in glycolysis and Krebs cycle.

• FAD/FADH₂: Accepts 2 electrons + 2 H⁺, used in Krebs cycle and fatty acid oxidation.

Redox Potential

• Redox Potential: Measures an atom's tendency to gain or lose electrons (e.g., NAD⁺ has E°' = -0.32 ext{ V}).

• Energy Harvest Role: Electron carriers transfer electrons to the electron transport chain yielding ATP.

• Diagnostic Tools: Redox sensors in diagnostic tools utilize NAD⁺-dependent enzymes to monitor blood sugar levels.

Metabolic Pathways Overview

Definition

• Metabolic Pathway: A series of chemical reactions modifying a starting molecule to produce a final product.

• Sugar metabolism has opposing pathways—synthesis (anabolic) vs. breakdown (catabolic).

• Anabolic Pathways: Build polymers, consuming energy.

• Catabolic Pathways: Break down polymers, releasing energy.

Anabolic Reactions

• Definition: Energy-consuming reactions (endergonic) that synthesize complex molecules from simpler ones.

• Example: Dehydration synthesis, such as bone mineralization—minerals crystallizing on the extracellular matrix.

Catabolic Reactions

• Definition: Energy-releasing reactions (exergonic) breaking down complex molecules into simpler compounds.

• Hydrolysis: The addition of water to break chemical bonds (e.g., breakdown of hydrogen peroxide into water and oxygen).

Forms of Energy in Biological Systems

Energy Dynamics

• Energy exists in various forms; chemical bonds allow for transformation and exchange to perform cellular processes.

• Thermodynamics: The study of energy transfer involving physical matter (open systems can exchange matter and energy, closed systems only energy).

Types of Energy

• Kinetic Energy: Energy of moving objects; relates to molecular movement and heat.

• Potential Energy: Stored energy based on position; includes compressed springs and chemical bonds.

• Chemical Energy: Stored in food molecules and released during their breakdown for cellular energy needs.

Thermodynamics in Biological Systems

First Law of Thermodynamics

• Energy cannot be created or destroyed, only transformed or transferred within biological systems.

• Biological systems harness energy from sunlight or chemical bonds for work.

Second Law of Thermodynamics

• Energy transfers are inefficient—some energy is transformed into unusable heat, contributing to increased disorder (entropy).

• Greater entropy signifies higher disorder.

Energy Transformation in Cellular Respiration

• Energy transfers from glucose (chemical potential energy) to ATP.

• Inefficient conversion results in energy loss as heat, consistent with the second law of thermodynamics.

Gibbs Free Energy

Definition

• Gibbs Free Energy (G): Total energy available for work at constant temperature and pressure.

• Spontaneity:

  • If ext{ΔG} < 0 → process is spontaneous (exergonic)

  • If ext{ΔG} > 0 → process is non-spontaneous (endergonic)

  • If ext{ΔG} = 0 → process is at equilibrium.

Gibbs Free Energy Key Formulas

1. Basic Formula: ext{ΔG} = ext{ΔH} - T ext{ΔS} (free energy change at constant temperature and pressure).

2. Non-Standard Conditions: ext{ΔG} = ext{ΔG}° + RT ext{ln}(Q) (adjusts for physiological conditions).

3. Equilibrium Constant Relationship: ext{ΔG}° = -RT ext{ln}(K_{ ext{eq}}) (free energy and reaction completion relation).

Sample Problem

• Determine spontaneity for: ext{ATP} + ext{H}_2 ext{O} ightarrow ext{ADP} + ext{Pi}

  • ext{ΔH} = -20 ext{ kJ/mol}, ext{ΔS} = +34 ext{ J/mol·K}, T = 310 ext{ K}

  • Calculation:

  • Convert ext{ΔS} to kJ: 34 ext{ J/mol·K} = 0.034 ext{ kJ/mol·K}

  • ext{ΔG} = -20 - (310 imes 0.034) = -30.54 ext{ kJ/mol} (spontaneous).

  • ATP breakdown releases energy for cellular work.

Energy Coupling and Phosphorylation

Energy Coupling Mechanism

• Energy Coupling: Links exergonic (energy-releasing) reactions with endergonic (energy-consuming) reactions through intermediates like ATP.

Types of Phosphorylation

• Substrate-Level Phosphorylation: Directly transfers phosphate to ADP (e.g., glycolysis yields 2 ATP).

• Oxidative Phosphorylation: Uses proton gradient generated from electron transport (e.g., yields 26-28 ATP from glucose).

• Photophosphorylation: Occurs in plants, using light energy for ATP synthesis (not applicable to human cells).

• Efficiency: Roughly 40-60% of glucose energy captured as ATP; remainder dissipates as heat.

• Example: Creatine phosphate in muscle cells assists rapid ATP regeneration.

Types of Bioenergetic Reactions

Exergonic Reactions

• Characteristics:

  • Release energy spontaneously (ΔG < 0).

  • Commonly found in biological processes, particularly in cellular respiration catabolic reactions.

  • Involve bond breaking.

Endergonic Reactions

• Characteristics:

  • Require an input of energy (ΔG > 0) and are non-spontaneous.

  • Common in anabolic processes such as photosynthesis and DNA/protein synthesis.

Glycolysis Overview

Definition

• Glycolysis: Anaerobic breakdown of glucose (C₆H₁₂O₆) into two pyruvate molecules occurring in the cytosol.

• Significance: First stage of cellular respiration, universal in all cells.

Key Features

• Location: Cytosol (does not require mitochondria).

• Oxygen Requirement: Anaerobic process; does not require oxygen.

• Phases: Two main phases—Energy Investment Phase (uses ATP) and Energy Payoff Phase (yields ATP/NADH).

Glycolysis Net Equation

• ext{C}6 ext{H}{12} ext{O}6 + 2 ext{NAD}^+ + 2 ext{ADP} + 2 ext{Pi} ightarrow 2 ext{CH}3 ext{COCOO}^- ( ext{Pyruvate}) + 2 ext{NADH} + 2 ext{ATP} + 2 ext{H}^+ + 2 ext{H}_2 ext{O}

• Role in Metabolism: Provides rapid energy; either links to Krebs Cycle (aerobic) or fermentation (anaerobic).

• ATP Yield: Net gain of 2 ATP per glucose (4 produced, 2 utilized), and produces 2 NADH (electron carriers feeding into ETC).

Glycolysis Phases

Energy Investment Phase

• Step 1: Glucose → Glucose-6-Phosphate (G6P)

  • Enzyme: Hexokinase (or Glucokinase in the liver)

  • Uses: 1 ATP; ext{ΔG}° ≈ -16.7 ext{ kJ/mol} (spontaneous).

  • Traps glucose in the cell.

• Step 2: G6P → Fructose-6-Phosphate (F6P)

  • Enzyme: Phosphoglucose Isomerase

  • Reversible isomerization with a low energy barrier.

• Step 3: F6P → Fructose-1,6-Bisphosphate (F1,6BP)

  • Enzyme: Phosphofructokinase-1 (PFK-1) - Key regulator.

  • Uses: 1 ATP; ext{ΔG}° ≈ -14.2 ext{ kJ/mol} (irreversible, commits to glycolysis).

• Step 4: F1,6BP → Dihydroxyacetone Phosphate (DHAP) + Glyceraldehyde-3-Phosphate (G3P)

  • Enzyme: Aldolase

  • Cleavage into two 3-carbon molecules.

• Step 5: DHAP → G3P

  • Enzyme: Triose Phosphate Isomerase

  • Converts DHAP to G3P; pathways yield two G3P molecules.

• Net for Energy Investment Phase: -2 ATP, preparing for energy harvest.

Energy Pay-off Phase

• Step 6: G3P → 1,3-Bisphosphoglycerate (1,3BPG)

  • Enzyme: Glyceraldehyde-3-Phosphate Dehydrogenase

  • Oxidation; NAD⁺ → NADH; adds Pi (high-energy bond).

• Step 7: 1,3BPG → 3-Phosphoglycerate (3PG)

  • Enzyme: Phosphoglycerate Kinase

  • Substrate-level phosphorylation; produces 1 ATP per G3P (total 2 ATP).

• Step 8: 3PG → 2-Phosphoglycerate (2PG)

  • Enzyme: Phosphoglycerate Mutase

  • Rearrangement; reversible reaction.

• Step 9: 2PG → Phosphoenolpyruvate (PEP)

  • Enzyme: Enolase

  • Dehydration creating high-energy bond.

• Step 10: PEP → Pyruvate

  • Enzyme: Pyruvate Kinase

  • Substrate-level phosphorylation; produces 1 ATP per PEP (total 2 ATP); ext{ΔG}° ≈ -31.4 ext{ kJ/mol} (irreversible).

Summary of Glycolysis Steps

Citric Acid Cycle

Overview

• The Citric Acid Cycle (Krebs Cycle) is crucial for aerobic metabolism.

• Location: Occurs in the mitochondrial matrix.

• Function: Oxidizes Acetyl-CoA to CO₂ and generates NADH, FADH₂, and GTP/ATP.

• Supplies electrons for the Electron Transport Chain.

Reaction Equation

• ext{Acetyl-CoA} + 3 ext{NAD}^+ + ext{FAD} + ext{GDP} + ext{Pi} + 2 ext{H}2 ext{O} ightarrow 2 ext{CO}2 + 3 ext{NADH} + ext{FADH}_2 + ext{GTP} + ext{CoA}

Steps of Citric Acid Cycle

Oxidative Phosphorylation & Electron Transport Chain (ETC)

Definition

• Oxidative Phosphorylation: The final stage of cellular respiration, occurring on the inner mitochondrial membrane.

Function

• Uses electrons from NADH and FADH₂ to establish a proton (H⁺) gradient.

• The gradient drives ATP synthase, generating ATP.

Complexes of the ETC

Alternative Pathways in Metabolism

Necessity for Alternative Pathways

• Alternative pathways are essential when:

  • Oxygen levels are low (hypoxia).

  • ATP demand fluctuates.

  • Biosynthesis needs arise.

  • Cells must regenerate NAD⁺.

Fermentation

• Lactic Acid Fermentation (in humans, muscle cells, and red blood cells):

  • Reaction: Pyruvate → Lactate

  • Enzyme: Lactate dehydrogenase; NADH is converted back to NAD⁺.

  • Yield: Only 2 ATP (via glycolysis).

  • Occurs in conditions of high exercise, hypoxia, or cancer (Warburg effect).

• Alcohol Fermentation (in yeast/bacteria):

  • Reaction: Pyruvate → Ethanol + CO₂.

  • Application: Brewing and biotechnology.

Gluconeogenesis

• Purpose: Maintain blood glucose during fasting; primarily occurs in the liver, also the kidney.

• Substrates:

  • Lactate (Cori cycle), glycerol, amino acids.

• Process: Reverse glycolysis bypassing irreversible steps with enzymes:

  • Pyruvate carboxylase, PEP carboxykinase, Fructose-1,6-bisphosphatase, Glucose-6-phosphatase.

• Energy Cost: Consumes 6 ATP per glucose.

• Function: Provides fuel supply for the brain and red blood cells.

• Pentose Phosphate Pathway (PPP):

  • Purpose: Not primarily for ATP production.

  • Produces NADPH and Ribose-5-phosphate, crucial for fatty acid and cholesterol synthesis, antioxidant defense, and detoxification.

Enzymes

Definition and Function

• Enzymes: Biological catalysts accelerating chemical reactions by lowering activation energy, vital for life processes.

• Enzymes form enzyme-substrate complexes, promoting easier bond breaking and formation without impacting reaction spontaneity or free energy levels; they only reduce activation energy.

General Properties

• Enzymes automatically recycle and can catalyze multiple reactions without being consumed.

• Enzymes mainly consist of protein and facilitate swift biochemical reactions by optimizing substrate orientation and altering active site conditions.

• Enzymes can stabilize transition states and interrupt chemical bond formations to lower activation energy.

Enzyme Activity Regulation

Influencing Factors

• Rate of Reaction Determinants:

  • Quantity of enzyme and substrate availability.

• Environmental Influences: pH, temperature, and salt concentrations significantly affect enzyme activity.

Inhibition Mechanisms

• Competitive Inhibition: An inhibitor competes for the active site with the substrate.

• Non-competitive Inhibition: An inhibitor binds to an allosteric site, altering enzyme conformation and preventing substrate binding.

• Promoted Activity: Presence of activators can enhance enzyme activity.

Michaelis–Menten Kinetics

Reaction Model

• E + S
  ightleftharpoons ES
  ightarrow E + P

• Michaelis–Menten Equation: v = rac{V{ ext{max}}[S]}{Km + [S]}

Key Parameters:

• Vmax: Maximum rate when all enzymes are saturated; depends on enzyme concentration.

• Km (Michaelis Constant): Concentration of substrate at half Vmax; indicates affinity:

  • Low Km → high affinity

  • High Km → low affinity

Behavioral Regions

• When [S] << Km → first-order kinetics (rate dependent on [S]).

• When [S] >> Km → zero-order kinetics (rate independent of [S])

• When [S] = Km → v = rac{1}{2} V_{ ext{max}}

Graphical Representations

• Velocity vs. Substrate Concentration: Includes Lineweaver-Burk and Eadie-Hofstee plots highlighting enzyme saturation behavior.

Conclusion

• These notes encompass the critical concepts surrounding enzymes and metabolism, focusing on cellular processes, energy transformations, and vital biochemical pathways. Further studies should integrate these foundational principles with advanced practical applications in biological systems.